Drug delivery systems for photodynamic therapy

ABSTRACT

The invention is generally related to the field of photodynamic therapy by use of photosensitizers and stabilized formulations of the photosensitizers. These formulations may be used to deliver a photosensitizer as a pharmaceutical, agricultural, or industrial agent. The photosensitizer containing formulations and compositions of the invention comprise one or more block copolymers. Furthermore, the invention relates to processes for the production of, and application of, said formulations and compositions as photosensitizer drug delivery systems.

RELATED APPLICATION

[0001] This application claims benefit of priority from U.S. ProvisionalApplication No. 60/202,641, filed May 8, 2000, which is herebyincorporated in its entirety as if fully set forth.

TECHNICAL FIELD

[0002] The invention is generally related to the field of photodynamictherapy by use of photosensitizers and stabilized formulations of thephotosensitizers. These formulations may be used to deliver aphotosensitizer as a pharmaceutical, agricultural, or industrial agent.The photosensitizer containing formulations and compositions of theinvention comprise one or more block copolymers. Additionally theinvention relates to the inclusion of one or more solid supports in suchformulations and compositions and/or the deposition of such formulationsand compositions on one or more solid supports. The inclusion of solidsupports increases the ease of hydrating the formulation or composition,which improves the effectiveness of the formulations and compositions asdelivery vehicles for photosensitizers. Furthermore, the inventionrelates to processes for the production of, and application of, saidformulations and compositions as photosensitizer drug delivery systems.

BACKGROUND OF THE INVENTION

[0003] Conventional photodynamic therapy (PDT) generally involves theadministration of a photosensitizer drug or compound to a recipient,either locally or systemically, followed by irradiation with light thatis capable of being absorbed by the photosensitizer in the tissue ororgan to be treated. The mode of photosensitizer drug delivery is ofparamount importance. The drug not only has to be in a form suitable foradministration, but also in a form that can readily undergo cellularinternalization at the target site, preferably with some degree ofselectivity over normal tissues.

[0004] There are multiple means of delivering pharmaceutical agents.These range from simple intravenous injection of solutions, emulsions,liposomes and microspheres to complex implantable time-release carriers.Photofrin® (QLT PhotoTherapeutics Inc., Vancouver, B.C., Canada, QLT)has been delivered successfully as part of a simple aqueous solution.Such aqueous solutions may not be suitable for hydrophobicphotosensitizer drugs of interest that have a tetra- orpoly-pyrrole-based structure. These drugs have an inherent tendency toaggregate by molecular stacking, which can severely curtail subsequentphotosensitization processes (Siggel et al. J. Phys. Chem.100(12):2070-2075, December 1996). One approach for maintaining lipidsoluble (hydrophobic) drugs in non-aggregated form is to formulate themin a hydrophobic liposomal bilayer.

[0005] Liposomal formulations of some hydrophobic photosensitizingdrugs, such as benzoporphyrin derivative monoacid-A (BPD-MA,Verteporfin®, QLT, Vancouver, Canada) and zinc phthalocyanine(CIBA-Geigy Ltd., Basel, Switzerland) are known. The liposome in thecase of BPD-MA acts as a passive delivery agent, transferring thephotosensitizer to plasma lipoproteins, such as low density lipoproteins(LDL), immediately upon injection into the blood stream. The highersurface expression of LDL receptors in rapidly proliferating tissuesaffords a level of selectivity to localization of hydrophobic LDLassociated drugs at target sites for PDT. Though liposomal formulationshave been successfully used for BPD-MA, they have been foundunsatisfactory for other, newer photosensitizers developed for PDT interms of drug loading, formulation stability and in vivo drug delivery.These photosensitizers are hydrophobic in nature and have propertiesthat promote considerably greater molecular stacking interactions; thus,drug aggregation was found to take place even within the liposomalbilayer.

[0006] Biocompatible block copolymers are receiving increasingly widerusage in the pharmaceutical industry to enhance drug solubility andbioavailability (reviewed by Schmolka, Chapter 10, pp 189-214, in Tarcha(Ed.) Polymers for Controlled Drug Delivery, CRC Press, Boch Raton,Fla., 1991). This usage has included administration of a number ofhydrophobic anti-cancer drugs. In the field of PDT, drug delivery usinga two step conjugation of block copolymer N-(2-hydroxypropyl)methacrylamide (HPMA) to photosensitizer drug (Peterson et al. CancerRes. 56(17):3980-3985, 1996) and, additionally, to antibodies(Omelyanenko et al. Int, J. Cancer. 75:600-608, 1998) have beenconducted. HPMA conjugated to photosensitizer drugs, adriamycin or mesochlorin e₆ (Mce₆), and then to antibodies, for homing the drug to cancercells, were found to be more effective than without the antibodies(Omelyanenko et al. Supra).

[0007] In the field of PDT, there is a continuing need for a drugdelivery system that is simple, non-toxic, chemically inert, economicaland can easily be used for formulating different types ofphotosensitizers. Requirements for a photosensitizer formulation includenot only maintaining the drug in a relatively non-aggregated form, butalso to achieve effective delivery to target site. The end-productshould ideally have an extended shelf life (preferably as a solid stateformulation) and be easy to reconstitute for administration. To preventembolisms, particle size for a parenteral formulation must not exceed 1μm. In the event that the formulation should prove to be unstable toautoclaving or gamma-radiation, particle size must be less than 0.2 μmin order to allow filter sterilization. Other requirements for aparenteral formulation include that they are sterile, isotonic, containnon-toxic components (biodegradable or readily excreted) and havephysical and chemical stability. The end-product should ideally have anextended shelf life (preferably as a solid state formulation). Allformulations, whether parenteral or otherwise, must be easily hydratedor reconstituted and be stable prior to administration and displayeffective delivery and performance at the target site, preferably withselective localization over normal tissues.

SUMMARY OF THE INVENTION

[0008] The present invention provides compositions and methods for drugformulations, storage and delivery methods useful for photodynamictherapy (PDT) utilizing photosensitizer drugs and one or more blockcopolymers as carriers. It has been discovered that these copolymershave wide ranging properties and have the potential to address manyneeds and formulation requirements of photosensitizer drug deliverysystems. The copolymers are simple to use, non-toxic, chemically inert,economical, and can easily be used for formulating a wide range ofphotosensitizing drugs in a form that is readily taken up by the targetcells. It has also been discovered that incorporation of hydrophobicphotosensitizer drugs in block copolymers can be an effective techniquefor maintaining the drugs in a non-aggregated form by forming simplemicelle, emulsion or gel complexes. Additionally, it has been discoveredthat incorporation of hydratable solid-supports in such formulationsimproves their hydration.

[0009] The present invention also provides methods for photosensitizerdrug release in a form suitable for administration to subjectsundergoing photodynamic therapy. The invention further provides methodsof preparing the aforementioned block copolymer comprisingphotosensitizer formulations. These methods comprise combining aphotosensitizer and one or more block copolymers followed by conversioninto a solid form. The solid form formulation containing thephotosensitizer and block copolymer complex may remain as a solid or beoptionally hydrated with an aqueous solution for storage or application.The formulation, either before or after hydration, may be furtherformulated with other pharmaceutically acceptable agents; alternatively,the formulation may be further processed before use for purposes such assize reduction. Preferably, the solid form or hydrated formulation willbe separated into doses appropriate for administering an effectiveamount of the photosensitizer to a subject.

[0010] Furthermore, the invention provides compositions and methods forformulating a photosensitizer drug and block copolymer complex depositedon or encapsulated by a solid-support. Hydration of the complex resultsin a non-aggregated photosensitizer drug formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 is a graphical representation of in vitro cellular uptakeof the photosensitizer B-B3 using block copolymer and liposomalformulations. Uptake of a copolymer Pluronic® P123 formulation was veryrapid compared to the BPD-MA liposomal formulation. 50% uptake level wasobserved to be close to ‘zero’ incubation time, with uptake of B-B3peaking in about 20 min. In comparison, BPD-MA achieved saturation levelat 30 min, with 50% uptake in approximately 5 min.

[0012]FIG. 2 compares the effectiveness of liposomal and copolymerformulations of B-B3 in controlling joint inflammation in the MRL-lprmouse model using transcutaneous PDT. Mice receiving copolymer aloneexhibited arthritic symptoms similar to the untreated control. Theliposomal formulation of photosensitizer B-B3 showed improvedsuppression of the inflammation compared to controls in the earlystages. Relative to the controls and the liposomal formulation, the B-B3copolymer formulation was highly effective in controlling theinflammation as determined by the increase in ankle swelling.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[0013] Prior to setting forth the invention, it may be helpful to anunderstanding thereof to first set forth definitions of certain termsthat will be used hereinafter.

[0014] “Block copolymer” and “copolymer” refer to carriers and carrieragents comprising any variation of two or more covalently linked blocks.The copolymers may be symmetric or asymmetric, amphiphilic (containingboth hydrophilic and hydrophobic chemical groups), graft, or random. Theblocks are linked by any appropriate linkage, including, but not limitedto, —CH₂—, —O—, —NH—, carbonyl, ester, amide, and imide linkages. Thecarriers may or may not be charged, and preferably comprise two or threeblocks. Preferably, the copolymers are symmetric or non-symmetric typetriblock copolymers, which may be represented as A-B-A and A-B-A′,respectively.

[0015] The carriers of the invention include poloxamers, or“PEO-PPO-PEO”, which are symmetrical triblock copolymers ofpolyoxyethylene (PEO, EO) and polyoxypropylene (PPO, PO) denoted asPEO-PPO-PEO or (EO)_(n1)(PO)_(m)(EO)_(n2) orHO(C₂H₄O)_(a)(C₃H₆O)_(b)(C₂H₄O)_(c)H. These copolymers are commerciallyavailable and have been well characterized in the art. Examples are thepoloxamers sold under various trademarks, such as Pluronic® (BASF Corp.)or Synperonics® (ICI).

[0016] Also within the scope of the invention are amphiphilic copolymersas described in WO 99/18998 (or its corresponding U.S. patent, if any),which is hereby incorporated by reference in its entirety as if fullyset forth. Explicitly excluded from inclusion for use alone as a “blockcopolymer” or “copolymer” of the invention, however, is an amphiphilicpolymer of polystyrene sodium sulphonate and vinyl naphthalene when thephotosensitizer used in the invention is 5,10,15,20 tetrakis phenylporphyrin. This specific amphiphilic polymer may also be excluded frominclusion for use alone when other photosensitizers are used in theinvention. Thus 5,10,15,20 tetrakis phenyl porphyrin may be used in theinvention if other copolymers or other photosensitizers or medicamentsare used.

[0017] Another “block copolymer” that may be excluded from inclusion foruse alone as a copolymer of the invention is Pluronic® F68 whenhematoporphyrin derivatives are used as the medicament. Thus thispoloxamer may be used as part of formulations containing additionalagents, such as those for forming emulsions, but preferably notfluorocarbons such as FC43, PP11, and PP25.

[0018] In addition to copolymers, carriers and carrier agents of theinvention include lipid compounds capable of forming or being associatedwith liposomes. In applications of the invention relating to liposomepreparation, the associated or incorporated medicament is preferablylimited either to photosensitizers or the use of exosupports. Carriersof the invention may be in a “liquid form”, which includes any liquid orliquefied form of the carrier. Examples of the “liquid form” of carriersare the carriers dissolved in solution and the carrier in a liquefiedform, such as in melted or molten forms. Preferred dissolved forms areprepared by solubilizing copolymers in appropriate solvents, preferablyvolatile solvents.

[0019] After formulation with a medicament of interest, the carrier maybe converted to a “solid form” by removal of solvent or otherwisesolidification of the carrier. Solvent removal may be by any means knownin the art, including, but not limited to, spray drying, lyophilization,heating, and application of a vacuum. Solidification, especially ofcarriers in a liquefied form, may be by any means known in the art.These include, but are not limited to, cooling or hardening in thepresence of a medicament or solid carrier.

[0020] “Complex” and “complexes” refer to stable micellar, emulsion,gel, matrix or transition phases between the defined states formed whena block copolymer and a medicament or photosensitizer associate toresult in such forms. In some instances, formulation of such complexesrequires the presence of additional agents that participate in theformation of micellar, emulsion, gel, matrix or transition phasestructures in solution. Examples of such agents include oils or otherlipids. The complexes of the invention may optionally includepharmaceutically acceptable excipients. They may also include adjuvants.

[0021] “Green porphyrins” refer to porphyrin derivatives obtained byreacting a porphyrin nucleus with an alkyne in a Diels-Alder typereaction to obtain a mono-hydrobenzoporphyrin.

[0022] “Solid support” or “support” refers to solid material with whicha medicament (or photosensitizer) and carrier mixture may becomeassociated. In cases of the mixture being in a solvent system, theassociation predominantly occurs upon solvent removal. The solidmaterials of the invention are normally not soluble in a solvent systemsolubilizing the medicament (or photosensitizer) and carrier mixture. Ofcourse combinations of solid support materials may be used inassociation with any medicament/carrier mixture.

[0023] In another aspect of the invention, the carrier in a molten orother liquefied form acts as a “solvent” for hydrophobic medicamentssuch as some photosensitizers, thus obviating the need for solventremoval to prepare the medicament/carrier mixture. Such “solvent”carriers in their molten, melted, or other liquefied form may be readilycombined with a medicament of interest. Examples of particularlyexcellent combinations using a “solvent” carrier as solvent includepoloxamers or polyethylene glycols (PEGs) as the “solvent” carrier withphotosensitizers. The ability to avoid extraneous solvent use isadvantageous for ecological, health, safety, and disposalconsiderations. It is also beneficial in simplifying the processesinvolved (i.e. need for special precautions, handling and/orinstrumentation) in preparing the compositions of the invention.

[0024] The compositions and methods of the invention may also serve toprepare a medicament in a “non-aggregated” form defined as that in whicha medicament (i.e. photosensitizer) does not exhibit sufficient strongintermolecular interactions with other medicament molecules to result insignificant aggregation.

[0025] The present invention provides compositions and methods for drugformulation as well as delivery methods useful for photodynamic therapyutilizing photosensitizers. Preferably, such compositions and methodscomprise one or more of block copolymers as the carrier to address theneeds described above. The photosensitizer and copolymer formulations ofthe invention include photosensitizer carrier compositions.

[0026] For example, one aspect of the present invention provides acomposition for formulating photosensitizers. This composition comprisesa photosensitizer drug and one or more block copolymers capable offorming complexes with the drug.

[0027] Another aspect of the present invention provides a method forformulating a photosensitizer comprising a) combining thephotosensitizer with one or more desired block copolymers in liquidform, and b) solidifying, optionally by drying, the mixture to produce acomplex of photosensitizer and block copolymer. The complex may then besubsequently hydrated with an aqueous solution to formphotosensitizer-carrier complexes, which may be administered in aneffective amount to a subject undergoing photodynamic therapy.

[0028] The compositions and methods of the present invention furtherinclude administration of simple formulations of photosensitizercompounds for recipients undergoing PDT treatment. The followingdescribes the photosensitizers, methods of administration, compositions,formulations and storage and handling of the present invention.Experimental data are also presented and described.

[0029] A. Photosensitizers

[0030] The invention may be practiced with a variety of synthetic andnaturally occurring pyrrole based photosensitizers, this includespro-drugs such as 5-aminolevulinic acid, porphyrins and porphyrinderivatives e.g. chlorins, bacteriochlorins, isobacteriochlorins,phthalocyanine and naphthalocyanines and other tetra- andpoly-macrocyclic compounds, and related compounds (e.g.pyropheophorbides, sapphyrins and texaphyrins) and metal complexes (suchas, but not limited by, tin, aluminum, zinc, lutetium).Tetrahydrochlorins, purpurins, porphycenes, and phenothiaziniums arealso within the scope of the invention.

[0031] Particularly preferred photosensitizers include green porphyrinssuch as BPD-MA, EA6 and B3. Generally, any polypyrrolic macrocyclicphotosensitive compound that is hydrophobic can be used in theinvention. Examples of these and other photosensitizers for use in thepresent invention include, but are not limited to, angelicins, somebiological macromolecules such as lipofuscin; photosystem II reactioncenters; and D1-D2-cyt b-559 photosystem II reaction centers,chalcogenapyrillium dyes, chlorins, chlorophylls, coumarins, cyanines,ceratin DNA and related compounds such as adenosine; cytosine;2′-deoxyguanosine-5′-monophosphate; deoxyribonucleic acid; guanine;4-thiouridine; 2′-thymidine 5′-monophosphate;thymidylyl(3′-5′)-2′-deoxyadenosine;thymidylyl(3′-5′)-2′-deoxyguanosine; thymine; and uracil, certain drugssuch as adriamycin; afloqualone; amodiaquine dihydrochloride;chloroquine diphosphate; chiorpromazine hydrochloride; daunomycin;daunomycinone; 5-iminodaunomycin; doxycycline; furosemide; gilvocarcinM; gilvocarcin V; hydroxychloroquine sulfate; lumidoxycycline;mefloquine hydrochloride; mequitazine; merbromin (mercurochrome);primaquine diphosphate; quinacrine dihydrochloride; quinine sulfate; andtetracycline hydrochloride, certain flavins and related compounds suchas alloxazine; flavin mononucleotide; 3-hydroxyflavone; limichrome;limiflavin; 6-methylalloxazine; 7-methylalloxazine; 8-methylalloxazine;9-methylalloxazine; 1-methyl limichrome; methyl-2-methoxybenzoate;5-nitrosalicyclic acid; proflavine; and riboflavin, fullerenes,metalloporphyrins, metallophthalocyanines, methylene blue derivatives,naphthalimides, naphthalocyanines, certain natural compounds such asbis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione;4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one; N-formylkynurenine;kynurenic acid; kynurenine; 3-hydroxykynurenine; DL-3-hydroxykynurenine;sanguinarine; berberine; carmane; and 5,7,9(11),22-ergostatetraene-3β-ol, nile blue derivatives, NSAIDs (nonsteroidal anti-inflammatorydrugs), perylenequinones, phenols, pheophorbides, pheophytins,photosensitizer dimers and conjugates, phthalocyanines, porphycenes,porphyrins, psoralens, purpurins, quinones, retinoids, rhodamines,thiophenes, verdins, vitamins and xanthene dyes (Redmond and Gamlin,Photochem. Photobiol., 70(4):391-475 (1999)).

[0032] Exemplary angelicins include 3-aceto-angelicin; angelicin;3,4′-dimethyl angelicin; 4,4′-dimethyl angelicin; 4,5′-dimethylangelicin; 6,4′-dimethyl angelicin; 6,4-dimethyl angelicin;4,4′,5′-trimethyl angelicin; 4,4′,5′-trimethyl-1′-thioangelicin;4,6,4′-trimethyl-1′-thioangelicin; 4,6,4′-trimethyl angelicin;4,6,5′-trimethyl-1′-thioangelicin; 6,4,4′-trimethyl angelicin;6,4′,5′-trimethyl angelicin; 4,6,4′,5′-tetramethyl-1′-thioangelicin; and4,6,4′,5′-tetramethyl angelicin. Exemplary chalcogenapyrillium dyesinclude pyrilium perchlorate,4,4′-(1,3-propenyl)-bis[2,6-di(1,1-dimethylethyl)]-; pyriliumperchlorate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)selenopyran-4-ylidene]-3-propenyl-;pyrilium hexofluoro phosphate,2,6-bis-(1,1-dimethyl-ethyl)-selenopyran-4-ylidene]-3-propenyl-;pyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-selenopyran-4-ylidene]-3-propenyl-; pyriliumperchlorate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl-;pyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl-;pyrilium perchlorate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)thiapyran-4-ylidene]-3-propenyl]-;selenopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)selenopyran-4-ylidene]-3-propenyl]-;selenopyrilium,2,6-bis(1,1-dimethylethyl)-4-[1-[2,6-bis(1,1-dimethylethyl)selenopyran-4-ylidene]-3-propenyl]-;selenopyrilium percheorate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl]-;selenopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl]-;selenopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[2-[2,6-bis(1,1-dimethyl-ethyl)selenopyran-4-ylidene]-4-(2-butenyl)]-;selenopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[2-[2,6-bis(1,1-dimethyl-ethyl)selenopyran-4-ylidene]-4-(2-pentenyl)]-;telluropyrilium tetrafluoroborate,2,6-bis(1,1-dimethylethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)-telluropyran-4-ylidene]-3-propenyl]-;telluropyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl]-;telluropyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]ethyl-;telluropyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)-telluropyran-4-ylidene]methyl-;thiopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)thiopyran-4-ylidene]-3-propenyl]-;thiopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)selenopyran-4-ylidene]-3-propenyl]-;and thiopyrilium hexofluoro phosphate,2,6-bis(1,1-dimethyl-ethyl)-4-[1-[2,6-bis(1,1-dimethyl-ethyl)telluropyran-4-ylidene]-3-propenyl]-.Exemplary chlorins dyes include 5-azachlorin dimethyl ester derivative;5,10,15,20-tetrakis-(m-hydroxyphenyl) bacteriochlorin; benzoporphyrinderivative monoacid ring A; benzoporphyrin derivative monoacid ring-A;porphine-2,18-dipropanoic acid,7-[2-dimethyl-amino)-2-oxoethyl]-8-ethylidene-7,8-dihydro-3,7,12,17-tetramethyl,dimethylester; porphine-2,18-dipropanoic acid,7-[2-dimethyl-amino)-2-oxoethyl]-8-ethylidene-8-ethyl-7,8-dihydro-3,7,12,17-tetramethyl,dimethylester Z; porphine-2,18-dipropanoic acid,7-[2-dimethyl-amino)-2-oxoethyl]-8-ethylidene-8-ethyl-7,8-dihydro-3,7,12,17-tetramethyl,dimethylester Z ECHL; porphine-2,18-dipropanoic acid,7-[2-dimethyl-amino)-2-oxoethyl]-8-ethylidene-8-n-heptyl-7,8-dihydro-3,7,12,17-tetramethyl,dimethylester Z; tin (II) porphine-2,18-dipropanoic acid,7-[2-(dimethylamino-2-oxoethyl]-8-ethylidene-8-n-heptyl-7,8-dihydro-3,7,12,17-tetramethyl,dimethylester Z; chlorin e₆; chlorin e₆ dimethyl ester; chlorin e₆ k₃;chlorin e₆ monomethyl ester; chlorin e₆ Na₃; chlorin p₆; chlorinp₆-trimethylester; chlorin derivative zinc (II)porphine-2,18-dipropanoic acid,7-[2-(dimethylamino)-2-oxoethyl]-8-ethylidene-8-n-heptyl-7,8-dihydro-3,7,12,17-tetramethyl,dimethylester Z; 13¹-deoxy-20-formyl-vic-dihydroxy-bacteriochlorindi-tert-butyl aspartate; 13¹-deoxy-20-formyl-4-keto-bacteriochlorindi-tert-butyl aspartate; di-L-aspartyl chlorin e₆; mesochlorin;5,10,15,20-tetrakis-(m-hydroxyphenyl) chlorin;meta-(tetrahydroxyphenyl)chlorin;methyl-13¹-deoxy-20-formyl-4-keto-bacteriochlorin; mono-L-aspartylchlorin e_(6;) photoprotoporphyrin IX dimethyl ester; phycocyanobilindimethyl ester; protochlorophyllide a; tin (IV) chlorin e₆; tin chlorine₆; tin L-aspartyl chlorin e₆; tin octaethyl-benzochlorin; tin (IV)chlorin; zinc chlorin e₆; and zinc L-aspartyl chlorin e₆.

[0033] Exemplary chlorophylls dyes include chlorophyll a; chlorophyll b;oil soluble chlorophyll; bacteriochlorophyll a; bacteriochlorophyll b;bacteriochlorophyll c; bacteriochlorophyll d; protochlorophyll;protochlorophyll a; amphiphilic chlorophyll derivative 1; andamphiphilic chlorophyll derivative 2.

[0034] Exemplary coumarins include 3-benzoyl-7-methoxycoumarin;7-diethylamino-3-thenoylcoumarin; 5,7-dimethoxy-3-(1-naphthoyl)coumarin; 6-methylcoumarin; 2H-selenolo[3,2-g] [1] benzopyran-2-one;2H-selenolo[3,2-g] [1] benzothiopyran-2-one; 7H-selenolo[3,2-g] [1]benzoseleno-pyran-7-one; 7H-selenopyrano[3,2-f] [1] benzofuran-7-one;7H-selenopyrano[3,2-f] [1] benzo-thiophene-7-one; 2H-thienol[3,2-g] [1]benzopyran-2-one; 7H-thienol[3,2-g] [1] benzothiopyran-7-one;7H-thiopyrano[3,2-f] [1] benzofuran-7-one; coal tar mixture; khellin; RG708; RG277; and visnagin.

[0035] Exemplary cyanines include benzoselenazole dye; benzoxazole dye;1,1′-diethyloxacarbocyanine; 1,1′-diethyloxadicarbocyanine;1,1′-diethylthiacarbocyanine; 3,3′-dialkylthiacarbocyanines (n=2-18);3,3′-diethylthiacarbocyanine iodide; 3,3′-dihexylselenacarbocyanine;kryptocyanine; MC540 benzoxazole derivative; MC540 quinoline derivative;merocyanine 540; and meso-ethyl, 3,3′-dihexylselenacarbocyanine.

[0036] Exemplary fullerenes include C₆₀; C₇₀; C₇₆; dihydro-fullerene;1,9-(4-hydroxy-cyclohexano)-buckminster-fullerene;[1-methyl-succinate-4-methyl-cyclohexadiene-2,3]-buckminster-fullerene;and tetrahydro fullerene.

[0037] Exemplary metalloporphyrins include cadmium (II) chlorotexaphyrinnitrate; cadmium (II) meso-diphenyl tetrabenzoporphyrin; cadmiummeso-tetra-(4-N-methylpyridyl)-porphine; cadmium (II) texaphyrin;cadmium (II) texaphyrin nitrate; cobaltmeso-tetra-(4-N-methylpyridyl)-porphine; cobalt (II)meso(4-sulfonatophenyl)-porphine; copper hematoporphyrin; coppermeso-tetra-(4-N-methylpyridyl)-porphine; copper (II)meso(4-sulfonatophenyl)-porphine; Europium (III) dimethyltexaphyrindihydroxide; gallium tetraphenylporphyrin; ironmeso-tetra(4-N-methylpyridyl)-porphine; lutetium (III)tetra(N-methyl-3-pyridyl)-porphyrin chloride; magnesium (II)meso-diphenyl tetrabenzoporphyrin; magnesium tetrabenzoporphyrin;magnesium tetrapbenylporphyrin; magnesium (II)meso(4-sulfonatophenyl)-porphine; magnesium (II) texaphyrin hydroxidemetalloporphyrin; magnesium meso-tetra-(4-N-methylpyridyl)-porphine;manganese meso-tetra-(4-N-methylpyridyl)-porphine; nickelmeso-tetra(4-N-methylpyridyl)-porphine; nickel (II)meso-tetra(4-sulfonatophenyl)-porphine; palladium (II)meso-tetra-(4-N-methylpyridyl)-porphine; palladiummeso-tetra-(4-N-methylpyridyl)-porphine; palladium tetraphenylporphyrin;palladium (II) meso(4-sulfonatophenyl)-porphine; platinum (II)meso(4-sulfonatophenyl)-porphine; samarium (II) dimethyltexaphyrindihydroxide; silver (II) meso(4-sulfonatophenyl)-porphine; tin (IV)protoporphyrin; tin meso-tetra-(4-N-methylpyridyl)-porphine; tinmeso-tetra(4-sulfonatophenyl)-porphine; tin (IV)tetrakis(4-sulfonatophenyl) porphyrin dichloride; zinc (II)15-aza-3,7,12,18-tetramethyl-porphyrinato-13,17-diyl-dipropionicacid-dimethylester; zinc (II) chlorotexaphyrin chloride; zinccoproporphyrin III; zinc (II)2,11,20,30-tetra-(1,1-dimethyl-ethyl)tetranaphtho(2,3-b:2′,3′-g:2″3″-1:2′″3′″-q)porphyrazine;zinc (II)2-(3-pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethylethyl)trinaphtho[2′,3′-g:2″3″1::2′″,3′″-q]porphyrazine; zinc (II)2,18-bis-(3-pyridyloxy)dibenzo[b,1]-10,26-di(1,1-dimethyl-ethyl)dinaphtho[2′,3′-g:2′″,3′″-q]porphyrazine;zinc (II)2,9-bis-(3-pyridyloxy)dibenzo[b,g]-17,26-di(1,1-dimethyl-ethyl)dinaphtho[2″,3″-1:2′″,3′″-q]porphyrazine;zinc (II) 2,9,16-tris-(3-pyridyloxy)tribenzo[b,g,1]-24=(1,1-dimethyl-ethyl)naphtho[2′″,3′″-q]porphyrazine;zinc (II) 2,3-bis-(3-pyridyloxy)benzo[b]-10,19,28-tri(1.1-dimethyl-ethyl)trinaphtho[2′,3′-g:2″,3″1:2′″,3′″-q]porphyrazine;zinc (II) 2,3,18,19-tetrakis-(3-pyridyloxy)dibenzo[b,1]-10,26-di(1,1-dimethyl-ethyl)trinaphtho[2′,3′-g:2′″,3′″-q]porphyrazine;zinc (II) 2,3,9,10-tetrakis-(3-pyridyloxy)dibenzo[b,g]-17,26-di(1,1-dimethyl-ethyl)dinaphtho[2″,3″-1:2′″,3′″-q]porphyrazine;zinc (II)2,3,9,10,16,17-hexakis-(3-pyridyloxy)tribenzo[b,g,1]-24-(1,1-dimethyl-ethyl)naphtho[2′″,3′″-q]porphyrazine;zinc (II)2-(3-N-methyl)pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethyl-ethyl)trinaphtho[2′,3′-g:2″,3″1:2′″,3′″-q]porphyrazinemonoiodide; zinc (II)2,18-bis-(3-(N-methyl)pyridyloxy)dibenzo[b,1]-10,26-di(1,1-dimethylethyl)dinaphtho[2′,3′-g:2′″,3′″-q]porphyrazinediiodide; zinc (II)2,9-bis-(3-(N-methyl)pyridyloxy)dibenzo[b,g]-17,26-di(1,1-dimethylethyl)dinaphtho[2″,3″-1:2′″,3′″-q]porphyrazinediiodide; zinc (II)2,9,16-tris-(3-(N-methyl-pyridyloxy)tribenzo[b,g,1]-24-(1,1-dimethylethyl)naphtho[2′″,3′″-q]porphyrazinetriiodide; zinc (II)2,3-bis-(3-(N-methyl)pyridyloxy)benzo[b]-10,19,28-tri(1,1-dimethylethyl)trinaphtho[2′,3′-g:2″,3″-1:2′″,3′″-q]porphyrazinediiodide; zinc (II)2,3,18,19-tetrakis-(3-(N-methyl)pyridyloxy)dibenzo[b,1]-10,26-di(1,1-dimethyl)dinaphtho[2′,3′-g:2′″,3′″-q]porphyrazinetetraiodide; zinc (II)2,3,9,10-tetrakis-(3-(N-methyl)pyridyloxy)dibenzo[g,g]-17,26-di(1,1-dimethylethyl)dinaphtho[2″,3″-1:2′″,3′″-q]porphyrazinetetraiodide; zinc (II)2,3,9,10,16,17-hexakis-(3-(N-methyl)pyridyloxy)tribenzo[b,g,1]-24-(1,1-dimethylethyl)naphtho[2′″,3′″-q]porphyrazinehexaiodide; zinc (II) meso-diphenyl tetrabenzoporphyrin; zinc (II)meso-triphenyl tetrabenzoporphyrin; zinc (II)meso-tetrakis(2,6-dichloro-3-sulfonatophenyl) porphyrin; zinc (II)meso-tetra-(4-N-methylpyridyl)-porphine; zinc (II)5,10,15,20-meso-tetra(4-octyl-phenylpropynyl)-porphine; zinc porphyrinc; zinc protoporphyrin; zinc protoporphyrin IX; zinc (II)meso-triphenyl-tetrabenzoporphyrin; zinc tetrabenzoporphyrin; zinc (II)tetrabenzoporphyrin; zinc tetranaphthaloporphyrin; zinctetraphenylporphyrin; zinc (II) 5,10,15,20-tetraphenylporphyrin; zinc(II) meso (4-sulfonatophenyl)-porphine; and zinc (II) texaphyrinchloride.

[0038] Exemplary metallophthalocyanines include aluminummono-(6-carboxy-pentyl-amino-sulfonyl)-trisulfo-phthalocyanine; aluminumdi-(6-carboxy-pentyl-amino-sulfonyl)-trisulfophthalocyanine; aluminum(II) octa-n-butoxy phthalocyanine; aluminum phthalocyanine; aluminum(III) phthalocyanine disulfonate; aluminum phthalocyanine disulfonate;aluminum phthalocyanine disulfonate (cis isomer); aluminumphthalocyanine disulfonate (clinical prep.); aluminum phthalocyaninephthalimido-methyl sulfonate; aluminum phthalocyanine sulfonate;aluminum phthalocyanine trisulfonate; aluminum (III) phthalocyaninetrisulfonate; aluminum (III) phthalocyanine tetrasulfonate; aluminumphthalocyanine tetrasulfonate; chloroaluminum phthalocyanine;chloroaluminum phthalocyanine sulfonate; chloroaluminum phthalocyaninedisulfonate; chloroaluminum phthalocyanine tetrasulfonate;chloroaluminum-t-butyl-phthalocyanine; cobalt phthalocyanine sulfonate;copper phthalocyanine sulfonate; copper (II)tetra-carboxy-phthalocyanine; copper (II)-phthalocyanine; coppert-butyl-phthalocyanine; copper phthalocyanine sulfonate; copper (II)tetrakis-[methylene-thio[(dimethyl-amino)methylidyne]]phthalocyaninetetrachloride; dichlorosilicon phthalocyanine; gallium (III)octa-n-butoxy phthalocyanine; gallium (II) phthalocyanine disulfonate;gallium phthalocyanine disulfonate; gallium phthalocyaninetetrasulfonate-chloride; gallium (II) phthalocyanine tetrasulfonate;gallium phthalocyanine trisulfonate-chloride; gallium (II)phthalocyanine trisulfonate; GaPcS₁tBu₃; GaPcS₂tBu₂; GaPcS₃tBu₁;germanium (IV) octa-n-butoxy phthalocyanine; germanium phthalocyaninederivative; silicon phthalocyanine derivative; germanium (IV)phthalocyanine octakis-alkoxy-derivatives; iron phthalocyaninesulfonate; lead (II) 2,3,9,10,16,17,23,24-octakis(3,6-dioxaheptyloxy)phthalocyanine; magnesium t-butyl-phthalocyanine; nickel (II)2,3,9,10,16,17,23,24-octakis(3,6-dioxaheptyloxy) phthalocyanine;palladium (II) octa-n-butoxy phthalocyanine; palladium (II)tetra(t-butyl)-phthalocyanine; (diol) (t-butyl)₃-phthalocyanatopalladium(II); ruthenium(II)dipotassium[bis(triphenyl-phosphine-monosulphonate) phthalocyanine;silicon phthalocyanine bis(tri-n-hexyl-siloxy)-; silicon phthalocyaninebis(tri-phenyl-siloxy)-; HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₃)₂;HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₂CH₃)₂; SiPc[OSi(CH₃)₂(CH₂)₃N(CH₃)₂]₂;SiPc[OSi(CH₃)₂(CH₂)₃N(CH₂CH₃)(CH₂)₂N(CH₃)₂]₂; tin (IV) octa-n-butoxyphthalocyanine; vanadium phthalocyanine sulfonate; zinc (II)octa-n-butoxy phthalocyanine; zinc (II)2,3,9,10,16,17,23,24-octakis(2-ethoxy-ethoxy) phthalocyanine; zinc (II)2,3,9,10,16,17,23,24-octakis(3,6-dioxaheptyloxy) phthalocyanine; zinc(II) 1,4,8,11,15,18,22,25-octa-n-butoxy-phthalocyanine;zn(II)-phthalocyanine-octabutoxy; zn(II)-phthalocyanine; zincphthalocyanine; zinc (II) phthalocyanine; zinc phthalocyanine andperdeuterated zinc phthalocyanine; zinc (II) phthalocyanine disulfonate;zinc phthalocyanine disulfonate; zinc phthalocyanine sulfonate; zincphthalocyanine tetrabromo-; zinc (II) phthalocyanine tetra-t-butyl-;zinc (II) phthalocyanine tetra-(t-butyl)-; zinc phthalocyaninetetracarboxy-; zinc phthalocyanine tetrachloro-; zinc phthalocyaninetetrahydroxyl; zinc phthalocyanine tetraiodo-; zinc (II)tetrakis-(1,1-dimethyl-2-phthalimido)ethyl phthalocyanine; zinc (II)tetrakis-(1,1-dimethyl-2-amino)-ethyl-phthalocyanine; zinc (II)phthalocyanine tetrakis(1,1-dimethyl-2-trimethyl ammonium)ethyltetraiodide; zinc phthalocyanine tetrasulphonate; zinc phthalocyaninetetrasulfonate; zinc (II) phthalocyanine tetrasulfonate; zinc (II)phthalocyanine trisulfonate; zinc phthalocyanine trisulfonate; zinc (II)(t-butyl)₃-phthalocyanine diol; zinctetradibenzobarreleno-octabutoxy-phthalocyanine; zinc (II)2,9,16,23,-tetrakis-(3-(N-methyl)pyridyloxy)phthalocyanine tetraiodide;and zinc (II)2,3,9,10,16,17,23,24-octakis-(3-(N-methyl)pyridyloxy)phthalocyaninecomplex octaiodide; and zinc (II)2,3,9,10,16,17,23,24-octakis-(3-pyridyloxy)phthalocyanine.

[0039] Exemplary methylene blue derivatives include 1-methyl methyleneblue; 1,9-dimethyl methylene blue; methylene blue; methylene blue (16μM); methylene blue (14 μM); methylene violet; bromomethylene violet;4-iodomethylene violet;1,9-dimethyl-3-dimethyl-amino-7-diethyl-amino-phenothiazine; and1,9-dimethyl-3-diethylamino-7-dibutyl-amino-phenothiazine.

[0040] Exemplary naphthalimides blue derivatives includeN,N′-bis-(hydroperoxy-2methoxyethyl)-1,4,5,8-naphthaldiimide;N-(hydroperoxy-2-methoxyethyl)-1,8-naphthalimide; 1,8-naphthalimide;N,N′-bis(2,2-dimethoxyethyl)-1,4,5,8-naphthaldiimide; andN,N′-bis(2,2-dimethylpropyl)-1,4,5,8-naphthaldiimide.

[0041] Exemplary naphthalocyanines include aluminumt-butyl-chloronaphthalocyanine; silicon bis(dimethyloctadecylsiloxy)2,3-naphthalocyanine; silicon bis(dimethyloctadecylsiloxy)naphthalocyanine; silicon bis(dimethylthexylsiloxy)2,3-naphthalocyanine; silicon bis(dimethylthexylsiloxy)naphthalocyanine; silicon bis(t-butyldimethylsiloxy)2,3-naphthalocyanine; silicon bis(tert-butyldimethylsiloxy)naphthalocyanine; silicon bis(tri-n-hexylsiloxy) 2,3-naphthalocyanine;silicon bis(tri-n-hexylsiloxy) naphthalocyanine; siliconnaphthalocyanine; t-butylnaphthalocyanine; zinc (II) naphthalocyanine;zinc (II) tetraacetyl-amidonaphthalocyanine; zinc (II)tetraaminonaphthalocyanine; zinc (II) tetrabenzamidonaphthalocyanine;zinc (II) tetrahexylamidonaphthalocyanine; zinc (II)tetramethoxy-benzamidonaphthalocyanine; zinc (II)tetramethoxynaphthalocyanine; zinc naphthalocyanine tetrasulfonate; andzinc (II) tetradodecylamidonaphthalocyanine.

[0042] Exemplary nile blue derivatives include benzo[a]phenothiazinium,5-amino-9-diethylamino-; benzo[a]phenothiazinium,5-amino-9-diethylamino-6-iodo-; benzo[a]phenothiazinium,5-benzylamino-9-diethylamino-; benzo[a]phenoxazinium,5-amino-6,8-dibromo-9-ethylamino-; benzo[a]phenoxazinium,5-amino-6,8-diiodo-9-ethylamino-; benzo[a]phenoxazinium,5-amino-6-bromo-9-diethylamino-; benzo[a]phenoxazinium,5-amino-9-diethylamino-(nile blue A); benzo[a]phenoxazinium,5-amino-9-diethylamino-2,6-diiodo-; benzo[a]phenoxazinium,5-amino-9-diethylamino-2,-iodo; benzo[a]phenoxazinium,5-amino-9-diethylamino-6-iodo-; benzo[a]phenoxazinium,5-benzylamino-9-diethylamino-(nile blue 2B);5-ethylamino-9-diethylamino-benzo[a]phenoselenazinium chloride;5-ethylamino-9-diethyl-aminobenzo[a]phenothiazinium chloride; and5-ethylamino-9-diethyl-aminobenzo[a]phenoxazinium chloride.

[0043] Exemplary NSAIDs (nonsteroidal anti-inflammatory drugs) includebenoxaprofen; carprofen; carprofen dechlorinated (2-(2-carbazolyl)propionic acid); carprofen (3-chlorocarbazole); chlorobenoxaprofen;2,4-dichlorobenoxaprofen; cinoxacin; ciprofloxacin;decarboxy-ketoprofen; decarboxy-suprofen; decarboxy-benoxaprofen;decarboxy-tiaprofenic acid; enoxacin; fleroxacin; fleroxacin-N-oxide;flumequine; indoprofen; ketoprofen; lomelfloxacin;2-methyl-4-oxo-2H-1,2-benzothiazine-1,1-dioxide; N-demethyl fleroxacin;nabumetone; nalidixic acid; naproxen; norfloxacin; ofloxacin;pefloxacin; pipemidic acid; piroxicam; suprofen; and tiaprofenic acid.

[0044] Exemplary perylenequinones include hypericins such as hypericin;hypericin monobasic sodium salt; di-aluminum hypericin; di-copperhypericin; gadolinium hypericin; terbium hypericin, hypocrellins such asacetoxy hypocrellin A; acetoxy hypocrellin B; acetoxy iso-hypocrellin A;acetoxy iso-hypocrellin B; 3,10-bis[2-(2-aminoethylamino)ethanol]hypocrellin B; 3,10-bis[2-(2-aminoethoxy)ethanol] hypocrellin B;3,10-bis[4-(2-aminoethyl)morpholine] hypocrellin B; n-butylaminatedhypocrellin B; 3,10-bis(butylamine) hypocrellin B; 4,9-bis(butylamine)hypocrellin B; carboxylic acid hypocrellin B; cystamine-hypocrellin B;5-chloro hypocrellin A or 8-chloro hypocrellin A; 5-chloro hypocrellin Bor 8-chloro hypocrellin B; 8-chloro hypocrellin B; 8-chloro hypocrellinA or 5-chloro hypocrellin A; 8-chloro hypocrellin B or 5-chlorohypocrellin B; deacetylated aldehyde hypocrellin B; deacetylatedhypocrellin B; deacetylated hypocrellin A; deacylated, aldehydehypocrellin B; demethylated hypocrellin B; 5,8-dibromo hypocrellin A;5,8-dibromo hypocrellin B; 5,8-dibromo iso-hypocrellin B;5,8-dibromo[1,12-CBr═CMeCBr(COMe)] hypocrellin B;5,8-dibromo[1,12-CHBrC(═CH₂)CBr(COMe)] hypocrellin B;5,8-dibromo[1-CH₂COMe, 12-COCOCH₂Br—] hypocrellin B; 5,8-dichlorohypocrellin A; 5,8-dichloro hypocrellin B; 5,8-dichlorodeacytylatedhypocrellin B; 5,8-diiodo hypocrellin A; 5,8-diiodo hypocrellin B;5,8-diiodo[1,12-CH═CMeCH(COCH₂I₂)—] hypocrellin B;5,8-diiodo[1,12-CH₂C(CH₂I)═C(COMe)—] hypocrellin B; 2-(N,N-diethylamino)ethylaminated hypocrellin B;3,10-bis[2-(N,N-diethylamino)-ethylamine]hypocrellin B;4,9-bis[2-(N,N-diethyl-amino)-ethylamine] iso-hypocrellin B;dihydro-1,4-thiazine carboxylic acid hypocrellin B; dihydro-1,4-thiazinehypocrellin B; 2-(N,N-dimethylamino) propylamine hypocrellin B;dimethyl-1,3,5,8,10,12-hexamethoxy-4,9-perylenequinone-6,7-diacetate;dimethyl-5,8-dihydroxy-1,3,10,13-tetramethoxy-4,9-perylenequinone-6,7-diacetate;2,11-dione hypocrellin A; ethanolamine hypocrellin B; ethanolamineiso-hypocrellin B; ethylenediamine hypocrellin B; 11-hydroxy hypocrellinB or 2-hydroxy hypocrellin B; hypocrellin A; hypocrellin B;5-iodo[1,12-CH₂C(CH₂I)═C(COMe)—] hypocrellin B;8-iodo[1,12-CH₂C(CH₂I)═C(COMe)—] hypocrellin B; 9-methylaminoiso-hypocrellin B; 3,10-bis[2-(N,N-methylamino)propylamine]hypocrellinB; 4,9-bis(methylamine iso-hypocrellin B; 14-methylamine iso-hypocrellinB; 4-methylamine iso-hypocrellin B; methoxy hypocrellin A; methoxyhypocrellin B; methoxy iso-hypocrellin A; methoxy iso-hypocrellin B;methylamine hypocrellin B; 2-morpholino ethylaminated hypocrellin B;pentaacetoxy hypocrellin A; PQP derivative; tetraacetoxy hypocrellin B;5,8,15-tribromo hypocrellin B; calphostin C, Cercosporins such asacetoxy cercosporin; acetoxy iso-cercosporin; aminocercosporin;cercosporin; cercosporin+iso-cercosporin (1/1 molar);diaminocercosporin; dimethylcercosporin; 5,8-dithiophenol cercosporin;iso-cercosporin; methoxycercosporin; methoxy iso-cercosporin;methylcercosporin; noranhydrocercosporin; elsinochrome A; elsinochromeB; phleichrome; and rubellin A.

[0045] Exemplary phenols include 2-benzylphenol; 2,2′-dihydroxybiphenyl;2,5-dihydroxybiphenyl; 2-hydroxybiphenyl; 2-methoxybiphenyl; and4-hydroxybiphenyl.

[0046] Exemplary pheophorbides include pheophorbide a; methyl13¹-deoxy-20-formyl-7,8-vic-dihydro-bacterio-meso-pheophorbide a;methyl-2-(1-dodecyloxyethyl)-2-devinyl-pyropheophorbide a;methyl-2-(1-heptyl-oxyethyl)-2-devinyl-pyropheophorbide a;methyl-2-(1-hexyl-oxyethyl)-2-devinyl-pyropheophorbide a;methyl-2-(1-methoxy-ethyl)-2-devinyl-pyropheophorbide a;methyl-2-(1-pentyl-oxyethyl)-2-devinyl-pyropheophorbide a; magnesiummethyl bacteriopheophorbide d; methyl-bacteriopheophorbide d; andpheophorbide.

[0047] Exemplary pheophytins include bacteriopheophytin a;bacteriopheophytin b; bacteriopheophytin c; bacteriopheophytin d;10-hydroxy pheophytin a; pheophytin; pheophytin a; and protopheophytin.

[0048] Exemplary photosensitizer dimers and conjugates include aluminummono-(6-carboxy-pentyl-amino-sulfonyl)-trisulfophthalocyanine bovineserum albumin conjugate; dihematoporphyrin ether (ester);dihematoporphyrin ether; dihematoporphyrin ether (ester)-chlorin;hematoporphyrin-chlorin ester; hematoporphyrin-low density lipoproteinconjugate; hematoporphyrin-high density lipoprotein conjugate;porphine-2,7,18-tripropanoic acid,13,13′-(1,3-propanediyl)bis[3,8,12,17-tetramethyl]-;porphine-2,7,18-tripropanoic acid,13,13′-(1,11-undecanediyl)bis[3,8,12,17-tetramethyl]-;porphine-2,7,18-tripropanoic acid,13,13′-(1,6-hexanediyl)bis[3,8,12,17-tetramethyl]-; SnCe6-MAb conjugate1.7:1; SnCe6-MAb conjugate 1.7:1; SnCe6-MAb conjugate 6.8:1; SnCe6-MAbconjugate 11.2:1; SnCe6-MAb conjugate 18.9:1; SnCe6-dextran conjugate0.9:1; SnCe6-dextran conjugate 3.5:1; SnCe6-dextran conjugate 5.5:1;SnCe6-dextran conjugate 9.9:1; a-terthienyl-bovine serum albuminconjugate (12:1); α-terthienyl-bovine serum albumin conjugate (4:1); andtetraphenylporphine linked to 7-chloroquinoline.

[0049] Exemplary phthalocyanines include (diol)(t-butyl)₃-phthalocyanine; (t-butyl)₄-phthalocyanine;cis-octabutoxy-dibenzo-dinaphtho-porphyrazine;trans-octabutoxy-dibenzo-dinaphtho-porphyrazine;2,3,9,10,16,17,23,24-octakis2-ethoxyethoxy) phthalocyanine;2,3,9,10,16,17,23,24-octakis(3,6-dioxaheptyloxy) phthalocyanine;octa-n-butoxy phthalocyanine; phthalocyanine; phthalocyanine sulfonate;phthalocyanine tetrasulphonate; phthalocyanine tetrasulfonate;t-butyl-phthalocyanine; tetra-t-butyl phthalocyanine; andtetradibenzobarreleno-octabutoxy-phthalocyanine.

[0050] Exemplary porphycenes include 2,3-(2³-carboxy-2⁴-methoxycarbonylbenzo)-7,12,17-tris(2-methoxyethyl) porphycene;2-(2-hydroxyethyl)-7,12,17-tri(2-methoxyethyl) porphycene;2-(2-hydroxyethyl)-7,12,17-tri-n-propyl-porphycene;2-(2-methoxyethyl)-7,12,17-tri-n-propyl-porphycene;2,7,12,17-tetrakis(2-methoxyethyl) porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-hydroxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-methoxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-n-hexyloxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-acetoxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-caproyloxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-pelargonyloxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-stearoyloxy-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-(N-t-butoxycarbonylglycinoxy)porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-[4-((β-apo-7-carotenyl)benzoyloxyl-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-amino-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-acetamido-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-glutaramido-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-(methyl-glutaramido)-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-9-(glutarimido)-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-3-(N,N-dimethylaminomethyl)-porphycene;2,7,12,17-tetrakis(2-methoxyethyl)-3-(N,N-dimethylaminomethyl)-porphycenehydrochloride; 2,7,12,17-tetrakis(2-ethoxyethyl)-porphycene;2,7,12,17-tetra-n-propyl-porphycene;2,7,12,17-tetra-n-propyl-9-hydroxy-porphycene;2,7,12,17-tetra-n-propyl-9-methoxy-porphycene;2,7,12,17-tetra-n-propyl-9-acetoxy porphycene;2,7,12,17-tetra-n-propyl-9-(t-butyl glutaroxy)-porphycene;2,7,12,17-tetra-n-propyl-9-(N-t-butoxycarbonylglycinoxy)-porphycene;2,7,12,17-tetra-n-propyl-9-(4-N-t-butoxy-carbonyl-butyroxy)-porphycene;2,7,12,17-tetra-n-propyl-9-amino-porphycene;2,7,12,17-tetra-n-propyl-9-acetamido-porphycene;2,7,12,17-tetra-n-propyl-9-glutaramido-porphycene;2,7,12,17-tetra-n-propyl-9-(methyl glutaramido)-porphycene;2,7,12,17-tetra-n-propyl-3-(N,N-dimethylaminomethyl) porphycene;2,7,12,17-tetra-n-propyl-9,10-benzo porphycene;2,7,12,17-tetra-n-propyl-9-p-benzoyl carboxy-porphycene;2,7,12,17-tetra-n-propyl-porphycene;2,7,12,17-tetra-t-butyl-3,6;13,16-dibenzo-porphycene;2,7-bis(2-hydroxyethyl)-12,17-di-n-propyl-porphycene;2,7-bis(2-methoxyethyl)-12,17-di-n-propyl-porphycene; and porphycene.

[0051] Exemplary porphyrins include 5-azaprotoporphyrin dimethylester;bis-porphyrin; coproporphyrin III; coproporphyrin III tetramethylester;deuteroporphyrin; deuteroporphyrin IX dimethylester;diformyldeuteroporphyrin IX dimethylester; dodecaphenylporphyrin;hematoporphyrin; hematoporphyrin (8 μM); hematoporphyrin (400 μM);hematoporphyrin (3 μM); hematoporphyrin (18 μM); hematoporphyrin (30μM); hematoporphyrin (67 μM); hematoporphyrin (150 μM); hematoporphyrinIX; hematoporphyrin monomer; hematoporphyrin dimer; hematoporphyrinderivative; hematoporphyrin derivative (6 μM); hematoporphyrinderivative (200 μM); hematoporphyrin derivative A (20 μM);hematoporphyrin IX dihydrochloride; hematoporphyrin dihydrochloride;hematoporphyrin IX dimethylester; haematoporphyrin IX dimethylester;mesoporphyrin dimethylester; mesoporphyrin IX dimethylester;monoformyl-monovinyl-deuteroporphyrin IX dimethylester;monohydroxyethylvinyl deuteroporphyrin;5,10,15,20-tetra(o-hydroxyphenyl) porphyrin;5,10,15,20-tetra(m-hydroxyphenyl) porphyrin;5,10,15,20-tetrakis-(m-hydroxyphenyl) porphyrin;5,10,15,20-tetra(p-hydroxyphenyl) porphyrin; 5,10,15,20-tetrakis(3-methoxyphenyl) porphyrin; 5,10,15,20-tetrakis (3,4-dimethoxyphenyl)porphyrin; 5,10,15,20-tetrakis (3,5-dimethoxyphenyl) porphyrin;5,10,15,20-tetrakis (3,4,5-trimethoxyphenyl) porphyrin;2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetraphenylporphyrin;Photofrin®; Photofrin II; porphyrin c; protoporphyrin; protoporphyrinIX; protoporphyrin dimethylester; protoporphyrin IX dimethylester;protoporphyrin propylaminoethylformamide iodide; protoporphyrinN,N-dimethylaminopropylformamide; protoporphyrinpropylaminopropylformamide iodide; protoporphyrin butylformamide;protoporphyrin N,N-dimethylamino-formamide; protoporphyrin formamide;sapphyrin 13,12,13,22-tetraethyl-2,7,18,23 tetramethylsapphyrin-8,17-dipropanol; sapphyrin 23,12,13,22-tetraethyl-2,7,18,23tetramethyl sapphyrin-8-monoglycoside; sapphyrin 3;meso-tetra-(4-N-carboxyphenyl)-porphine;tetra-(3-methoxyphenyl)-porphine;tetra-(3-methoxy-2,4-difluorophenyl)-porphine;5,10,15,20-tetrais(4-N-methylpyridyl) porphine;meso-tetra-(4-N-methylpyridyl)-porphine tetrachloride;meso-tetra(4-N-methylpyridyl)-porphine;meso-tetra-(3-N-methylpyridyl)-porphine;meso-tetra-(2-N-methylpyridyl)-porphine;tetra(4-N,N,N-trimethylanilinium) porphine;meso-tetra-(4-N,N,N″-trimethylamino-phenyl) porphine tetrachloride;tetranaphthaloporphyrin; 5,10,15,20-tetraphenylporphyrin;tetraphenylporphyrin; meso-tetra-(4-N-sulfonatophenyl)-porphine;tetraphenylporphine tetrasulfonate;meso-tetra(4-sulfonatophenyl)porphine; tetra(4-sulfonatophenyl)porphine;tetraphenylporphyrin sulfonate; meso-tetra(4-sulfonatophenyl)porphine;tetrakis (4-sulfonatophenyl)porphyrin;meso-tetra(4-sulfonatophenyl)porphine; meso(4-sulfonatophenyl)porphine;meso-tetra(4-sulfonatophenyl)porphine;tetrakis(4-sulfonatophenyl)porphyrin;meso-tetra(4-N-trimethylanilinium)-porphine; uroporphyrin; uroporphyrinI (17 μM); uroporphyrin IX; and uroporphyrin I (18 μM).

[0052] Exemplary psoralens include psoralen; 5-methoxypsoralen;8-methoxypsoralen; 5,8-dimethoxypsoralen; 3-carbethoxypsoralen;3-carbethoxy-pseudopsoralen; 8-hydroxypsoralen; pseudopsoralen;4,5′,8-trimethylpsoralen; allopsoralen; 3-aceto-allopsoralen;4,7-dimethyl-allopsoralen; 4,7,4′-trimethyl-allopsoralen;4,7,5′-trimethyl-allopsoralen; isopseudopsoralen;3-acetoisopseudopsoralen; 4,5′-dimethyl-isopseudopsoralen;5′,7-dimethyl-isopseudopsoralen; pseudoisopsoralen;3-acetopseudoisopsoralen; 3/4′,5′-trimethyl-aza-psoralen;4,4′,8-trimethyl-5′-amino-methylpsoralen;4,4′,8-trimethyl-phthalamyl-psoralen; 4,5′,8-trimethyl-4′-aminomethylpsoralen; 4,5′,8-trimethyl-bromopsoralen; 5-nitro-8-methoxy-psoralen;5′-acetyl-4,8-dimethyl-psoralen; 5′-aceto-8-methyl-psoralen; and5′-aceto-4,8-dimethyl-psoralen Exemplary purpurins includeoctaethylpurpurin; octaethylpurpurin zinc; oxidized octaethylpurpurin;reduced octaethylpurpurin; reduced octaethylpurpurin tin; purpurin 18;purpurin-18; purpurin-18-methyl ester; purpurin; tin ethyl etiopurpurinI; Zn(II) aetio-purpurin ethyl ester; and zinc etiopurpurin.

[0053] Exemplary quinones include 1-amino-4,5-dimethoxy anthraquinone;1,5-diamino-4,8-dimethoxy anthraquinone; 1,8-diamino-4,5-dimethoxyanthraquinone; 2,5-diamino-1,8-dihydroxy anthraquinone;2,7-diamino-1,8-dihydroxy anthraquinone; 4,5-diamino-1,8-dihydroxyanthraquinone; mono-methylated 4,5- or 2,7-diamino-1,8-dihydroxyanthraquinone; anthralin (keto form); anthralin; anthralin anion;1,8-dihydroxy anthraquinone; 1,8-dihydroxy anthraquinone (Chrysazin);1,2-dihydroxy anthraquinone; 1,2-dihydroxy anthraquinone (Alizarin);1,4-dihydroxy anthraquinone (Quinizarin); 2,6-dihydroxy anthraquinone;2,6-dihydroxy anthraquinone (Anthraflavin); 1-hydroxy anthraquinone(Erythroxy-anthraquinone); 2-hydroxy-anthraquinone;1,2,5,8-tetra-hydroxy anthraquinone (Quinalizarin);3-methyl-1,6,8-trihydroxy anthraquinone (Emodin); anthraquinone;anthraquinone-2-sulfonic acid; benzoquinone; tetramethyl benzoquinone;hydroquinone; chlorohydroquinone; resorcinol; and 4-chlororesorcinol.

[0054] Exemplary retinoids include all-trans retinal; C₁₇ aldehyde; C₂₂aldehyde; 11-cis retinal; 13-cis retinal; retinal; and retinalpalmitate.

[0055] Exemplary rhodamines include 4,5-dibromo-rhodamine methyl ester;4,5-dibromo-rhodamine n-butyl ester; rhodamine 101 methyl ester;rhodamine 123; rhodamine 6G; rhodamine 6G hexyl ester;tetrabromo-rhodamine 123; and tetramethyl-rhodamine ethyl ester.

[0056] Exemplary thiophenes include terthiophenes such as2,2′:5′,2″-terthiophene; 2,2′:5′,2″-terthiophene-5-carboxamide;2,2′:5′,2″-terthiophene-5-carboxylic acid;2,2′:5′,2″-terthiophene-5-L-serine ethyl ester;2,2′:5′,2″-terthiophene-5-N-isopropynyl-formamide;5-acetoxymethyl-2,2′:5′,2″-terthiophene;5-benzyl-2,2′:5′,2″-terthiophene-sulphide;5-benzyl-2,2′:5′,2″-terthiophene-sulfoxide;5-benzyl-2,2′:5′,2″-terthiophene-sulphone;5-bromo-2,2′:5′,2″-terthiophene;5-(butynyl-3′″-hydroxy)-2,2′:5′,2″-terthiophene;5-carboxyl-5″-trimethylsilyl-2,2′:5′,2″-terthiophene;5-cyano-2,2′:5′,2″-terthiophene; 5,5″-dibromo-2,2′:5′,2″-terthiophene;5-(1′″,1′″-dibromoethenyl)-2,2′:5′,2″-terthiophene;5,5″-dicyano-2,2′:5′,2″-terthiophene;5,5″-diformyl-2,2′:5′,2″-terthiophene;5-difluoromethyl-2,2′:5′,2″-terthiophene;5,5″-diiodo-2,2′:5′,2″-terthiophene;3,3″-dimethyl-2,2′:5′,2″-terthiophene;5,5″-dimethyl-2,2′:5′,2″-terthiophene;5-(3′″,3′″-dimethylacryloyloxymethyl)-2,2′:5′,2″-terthiophene;5,5″-di-(t-butyl)-2,2′:5′,2″-terthiophene;5,5″-dithiomethyl-2,2′:5′,2″-terthiophene;3′-ethoxy-2,2′:5′,2″-terthiophene; ethyl2,2′:5′,2″-terthiophene-5-carboxylic acid;5-formyl-2,2′:5′,2″-terthiophene;5-hydroxyethyl-2,2′:5′,2″-terthiophene;5-hydroxymethyl-2,2′:5′,2″-terthiophene; 5-iodo-2,2′:5′,2″-terthiophene;5-methoxy-2,2′:5′,2″-terthiophene; 3′-methoxy-2,2′:5′,2″-terthiophene;5-methyl-2,2′:5′,2″-terthiophene;5-(3′″-methyl-2′″-butenyl)-2,2′:5′,2″-terthiophene; methyl2,2′:5′,2″-terthiophene-5-[3′″-acrylate]; methyl2,2′:5′,2″-terthiophene-5-(3′″-propionate);N-allyl-2,2′:5′,2″-terthiophene-5-sulphonamide;N-benzyl-2,2′:5′,2″-terthiophene-5-sulphonamide;N-butyl-2,2′:5′,2″-terthiophene-5-sulphonamide;N,N-diethyl-2,2′:5′,2″-terthiophene-5-sulphonamide;3,3′,4′,3″-tetramethyl-2,2′:5′,2″-terthiophene;5-t-butyl-5″-trimethylsilyl-2,2′:5′,2″-terthiophene;3′-thiomethyl-2,2′:5′,2″-terthiophene;5-thiomethyl-2,2′:5′,2″-terthiophene;5-trimethylsilyl-2,2′:5′,2″-terthiophene, bithiophenes such as2,2′-bithiophene; 5-cyano-2,2′-bithiophene; 5-formyl-2,2′-bithiophene;5-phenyl-2,2′-bithiophene; 5-(propynyl)-2,2′-bithiophene;5-(hexynyl)-2,2′-bithiophene; 5-(octynyl)-2,2′-bithiophene;5-(butynyl-4″-hydroxy)-2,2′-bithiophene;5-(pentynyl-5″-hydroxy)-2,2′-bithiophene;5-(3″,4″-dihydroxybutynyl)-2,2′-bithiophene derivative;5-(ethoxybutynyl)-2,2′-bithiophene derivative, and misclaneousthiophenes such as 2,5-diphenylthiophene; 2,5-di(2-thienyl)furan;pyridine,2,6-bis(2-thienyl)-; pyridine, 2,6-bis(thienyl)-; thiophene,2-(1-naphthalenyl)-; thiophene, 2-(2-naphthalenyl)-; thiophene,2,2′-(1,2-phenylene)bis-; thiophene, 2,2′-(1,3-phenylene)bis-;thiophene, 2,2′-(1,4-phenylene)bis-; 2,2′:5′,2″:5″,2′″-quaterthiophene;α-quaterthienyl; α-tetrathiophene; α-pentathiophene; α-hexathiophene;and α-heptathiophene.

[0057] Exemplary verdins include copro (II) verdin trimethyl ester;deuteroverdin methyl ester; mesoverdin methyl ester; and zinc methylpyroverdin.

[0058] Exemplary vitamins include ergosterol (provitamin D2);hexamethyl-Co a Cob-dicyano-7-de(carboxymethyl)-7,8-didehydro-cobyrinate (Pyrocobester);pyrocobester; and vitamin D3.

[0059] Exemplary xanthene dyes include Eosin B(4′,5′-dibromo,2′,7′-dinitro-fluorescein, dianion); eosin Y; eosin Y(2′,4′,5′,7′-tetrabromo-fluorescein, dianion); eosin(2′,4′,5′,7′-tetrabromo-fluorescein, dianion); eosin(2′,4′,5′,7′-tetrabromo-fluorescein, dianion) methyl ester; eosin(2′,4′,5′,7′-tetrabromo-fluorescein, monoanion)p-isopropylbenzyl ester;eosin derivative (2′,7′-dibromo-fluorescein, dianion); eosin derivative(4′,5′-dibromo-fluorescein, dianion); eosin derivative(2′,7′-dichloro-fluorescein, dianion); eosin derivative(4′,5′-dichloro-fluorescein, dianion); eosin derivative(2′,7′-diiodo-fluorescein, dianion); eosin derivative(4′,5′-diiodo-fluorescein, dianion); eosin derivative(tribromo-fluorescein, dianion); eosin derivative(2′,4′,5′,7′-tetrachloro-fluorescein, dianion); eosin; eosindicetylpyridinium chloride ion pair; erythrosin B(2′,4′,5′,7′-tetraiodo-fluorescein, dianion); erythrosin; erythrosindianion; erythrosin B; fluorescein; fluorescein dianion; phloxin B(2′,4′,5′,7′-tetrabromo-3,4,5,6-tetrachloro-fluorescein, dianion);phloxin B (tetrachloro-tetrabromo-fluorescein); phloxine B; rose bengal(3,4,5,6-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein, dianion); rosebengal; rose bengal dianion; rose bengal O-methyl-methylester; rosebengal 6′-O-acetyl ethyl ester; rose bengal benzyl esterdiphenyl-diiodonium salt; rose bengal benzyl ester triethylammoniumsalt; rose bengal benzyl ester, 2,4,6,-triphenylpyrilium salt; rosebengal benzyl ester, benzyltriphenyl-phosphonium salt; rose bengalbenzyl ester, benzyltriphenyl phosphonium salt; rose bengal benzylester, diphenyl-iodonium salt; rose bengal benzyl ester,diphenyl-methylsulfonium salt; rose bengal benzyl ester,diphenyl-methyl-sulfonium salt; rose bengal benzyl ester,triethyl-ammonium salt; rose bengal benzyl ester, triphenyl pyrilium;rose bengal bis (triethyl-ammonium) salt)(3,4,5,6-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein, bis(triethyl-ammonium salt); rose bengal bis (triethyl-ammonium) salt; rosebengal bis(benzyl-triphenyl-phosphonium) salt(3,4,5,6-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein, bis(benzyl-triphenyl-phosphonium) salt); rose bengal bis(diphenyl-iodonium) salt(3,4,5,6-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein,bis(diphenyl-iodonium) salt); rose bengal di-cetyl-pyridinium chlorideion pair; rose bengal ethyl ester triethyl ammonium salt; rose bengalethyl ester triethyl ammonium salt; rose bengal ethyl ester; rose bengalmethyl ester; rose bengal octyl ester tri-n-butyl-ammonium salt RB; rosebengal, 6′-O-acetyl-, and ethyl ester.

[0060] In one embodiment the preferred compounds for formulating are thehighly hydrophobic tetrapyrrolic A and B-ring compounds, such as BPD-DA,-DB, -MA, and -MB. Most preferred are the B-ring compounds, BPD-MB,B-EA6, B-B3; the A-ring compounds BPD-MA, A-EA6 and A-B3; anddihydroxychlorins.

[0061] These compounds are porphyrin derivatives obtained by reacting aporphyrin nucleus with an alkyne in a Diels-Alder type reaction toobtain a monohydrobenzoporphyrin, and they are described in detail inthe issued U.S. Pat. No. 5,171,749, which is hereby incorporated in itsentirety by reference. Of course, combinations of photosensitizers mayalso be used. It is preferred that the absorption spectrum of thephotosensitizer be in the visible range, typically between 350 nm and1200 nm, more preferably between 400-900 nm, and even more preferablybetween 600-900 nm.

[0062] BPD-MA is described, for example, in U.S. Pat. No. 5,171,749; EA6and B3 are described in U.S. Ser. Nos. 09/088,524 and 08/918,840,respectively, all of which are incorporated herein by reference.Preferred green porphyrins have the basic structure:

[0063] where R⁴ is vinyl or 1-hydroxyethyl and R¹, R², and R³ are H oralkyl or substituted alkyl. BPD-MA has the structure shown in formula 1wherein R¹ and R² are methyl, R⁴ is vinyl and one of R³ is H and theother is methyl. EA6 is of formula 2 wherein R¹ and R² are methyl andboth R³ are 2-hydroxyethyl (i.e., the ethylene glycol esters). B3 is offormula 2 wherein R¹ is methyl, R² is H, and both R³ are methyl. In bothEA6 and B3, R⁴ is also vinyl.

[0064] The representations of BPD-MAC and BPD-MAD, which are thecomponents of Verteporfin, as well as illustrations of A and B ringforms of EA6 and B3, are as follows:

[0065] Related compounds of formulas 3 and 4 are also useful; ingeneral, R⁴ will be vinyl or 1-hydroxyethyl and R¹, R², and R³ are H oralkyl or substituted alkyl.

[0066] Optionally excluded from inclusion as a photosensitizer of theinvention, however, is 5,10,15,20 tetrakis phenyl porphyrin.

[0067] Dimeric forms of the green porphyrin and dimeric or multimericforms of green porphyrin/porphyrin combinations may also be used. Thedimers and oligomeric compounds of the invention can be prepared usingreactions analogous to those for dimerization and oligomerization ofporphyrins per se. The green porphyrins or green porphyrin/porphyrinlinkages can be made directly, or porphyrins may be coupled, followed bya Diels-Alder reaction of either or both terminal porphyrins to convertthem to the corresponding green porphyrins.

[0068] Other non-limiting examples of photosensitizers which may beuseful in the invention are photosensitizing Diels-Alder porphyrinderivatives, described in U.S. Pat. No. 5,308,608; porphyrin-likecompounds, described in U.S. Pat. Nos. 5,405,957, 5,512,675, and5,726,304; bacteriochlorophyll-A derivatives described in U.S. Pat. Nos.5,171,741 and 5,173,504; chlorins, isobacteriochlorins andbacteriochlorins, as described in U.S. Pat. No. 5,831,088;meso-monoiodo-substituted and meso substituted tripyrrane, described inU.S. Pat. No. 5,831,088; polypyrrolic macrocycles from meso-substitutedtripyrrane compounds, described in U.S. Pat. Nos. 5,703,230, 5,883,246,and 5,919,923; and ethylene glycol esters, described in U.S. Pat. No.5,929,105. All of the patents cited in this paragraph are herebyincorporated by reference as if fully set forth. Generally anyhydrophobic or hydrophilic photosensitizers, which absorb in theultra-violet, visible and infra-red spectroscopic ranges would be usefulfor practicing this invention.

[0069] Presently a number of photosensitizer drugs of interest arehydrophobic with a tetrapyrrole-based structure. These drugs have aninherent tendency to aggregate, which can severely curtailphotosensitization processes (Siggel et al. J. Phys. Chem.100(12):2070-2075, December 1996). For example, the synthetic pathwayfor BPD yields A and B ring intermediates in approximately equimolarquantities, which can be derivatized further. It was found that theA-ring derivatives, such as BPD-MA (Verteporfin), could easily beformulated for delivery using traditional means, whereas B-ringcompounds proved more difficult to formulate due to their tendency toundergo self-association.

[0070] In an additional aspect of the invention, the photosensitizers ofthe invention may be conjugated to various ligands that facilitatetargeting to tissues and cells before the photosensitizers areformulated with block copolymers. These ligands include those that arereceptor-specific as well as immunoglobulins and fragments thereof.Preferred ligands include antibodies in general and monoclonalantibodies, as well as immunologically reactive fragments thereofMoreover, the block copolymer may be conjugated to the ligands to whichthe photosensitizer binds to facilitate improved complexing ofnon-hydrophobic photosensitizers with the copolymer.

[0071] B. Block Copolymers

[0072] The formulations of the invention may be practiced with a varietyof carrier agents, including combinations of such agents. The preferredcarrier agents of the invention are symmetric and asymmetric blockcopolymers composed of two or more blocks. These can be amphiphilicrandom, graft, or block copolymers, either branched or linear which canbe biodegradable or otherwise excretable. The hydrophobe is the part ofthe copolymer that can interact with the photosensitizer. Examplesinclude, but are not limited to, homo- or hetero-polymers composed ofamino acids such as tryptophan, histidine, aspartate, or phenylalanine;pyridines, purines, or indoles; toluene, benzene and alkyl benzene,anthracene, or phenanthrene; and propylene glycol. The hydrophile can beselected from, but is not limited to, any of the following: polyethyleneglycol, polyetbylene oxide, poly amino acids, polycarboxylates andpolysulphonates. Blocks and/or monomers within the blocks are linked by,but not limited to groups such as —CH2—, —O—, —NH—, carbonyl, ester,amide and imine linkages. More preferred are the symmetric andasymmetric block polymers of the structure A-B-A and A-B-A′,respectively, where the ratio of hydrophilic to hydrophobic groups rangefrom 1:20 to 20:1. Most preferred are those that can form micellar/mixedmicelle suspensions, emulsions, gels or other stable complexes with thephotosensitizer of interest. Additional carriers of the inventioninclude lipid-containing compounds capable of forming or beingassociated with liposomes.

[0073] Where block copolymers are used, the copolymers are preferablywater-soluble triblock copolymers of composed of polyethylene oxide(PEO), and polypropylene oxide (PPO) denoted as PEO-PPO-PEO or(EO)n1(PO)m(EO)n2 or HO(C2H4O)a(C3H6O)b(C2H4O)cH (Schmolka, Supra;Alexandridis & Hatton, Colloids and Surfaces 96:1-46, 1995). Morepreferred are those where a and c are independently from 1-150 units andb ranges from 10-200 units with the overall molecular weight rangingfrom 1,000 to 50,000 daltons. Particularly preferred are those where aequals c and b ranges from 10-200 units.

[0074] Others examples of block copolymers that are useful for thisinvention are those where the central block is composed of otheramphiphilic, charged or uncharged monomeric groups which are likely tointeract more specifically with a photosensitizer of interest (Kataokaet al. J. Controlled Release 24:119-132, 1993). These moieties areselected depending on the properties (polarity, charge, aromaticcharacter, etc.) of the photosensitizer to be formulated.

[0075] Block copolymers that would be useful in this invention are ofthe non-toxic di-block, symmetric and non-symmetric triblock copolymersand dendrimer types. More preferable are the symmetrical triblockcopolymers, preferably those composed of PEO-PPO-PEO types of blockcopolymers, where the hydrophobic PPO provides the methyl groups thatare believed to interact with and stabilize the substance to besolubilized.

[0076] PEO confers water solubility to the copolymer, although thehydrogen bonding interactions of the ether oxygen with water moleculesprobably occurs along the entire copolymer. These copolymers areavailable from a number of commercial sources such as BASF Corporation(Pluronic®series) and ICI (Synperonic® series). In the numeric namingsystem for both the series, the last digit of the copolymer numbermultiplied by 10 gives the approximate percent molecular weight of thehydrophilic blocks (PEO). Poloxamers can be roughly divided into 3 maincategories, all of which can be useful for stabilizing and delivery ofdrug substances, namely emulsion forming, micelle forming, and watersoluble ones which form an extended network in solution. At higherconcentrations they have a tendency to undergo gel formation undercertain temperature conditions (Edsman et al. Eur J Pharm Sci. 6,105-112, 1998). Some of the important factors which determine poloxamercharacteristics and behavior in aqueous suspension are the molecularweight, PPO:PEO ratio, temperature conditions, concentration, andpresence of ionic materials. There is consequently a wide range ofcharacteristics in existing commercially available copolymers, which canbe exploited for formulation purposes, whether for merely monomerizationof hydrophobic photosensitizers or for controlled drug deliverypurposes. Additionally, alternative PEO-PPO-PEO polymers can be tailoredaccording to requirements of a particular drug substance e.g. molecularweight, PPO:PEO ratio, as well as administration route.

[0077] Another characteristic of the copolymers is their wetting ordetergent capacity which has been used to promote plasma membranepermeability of various drugs (Melik-Nubarov et al., FEBS Lett.5;446(1):194-198, 1999), and thereby increasing bioavailability of thedrugs. It has been shown that these copolymers can also act asimmunoadjuvants (Hunter et al. Aids Research and Human Retroviruses 10(Supplement 2): S95-S98, 1994) and could improve the benefits of aregime, for example if used in conjunction with PDT particularly forautoimmune disorders.

[0078] The present invention includes the observation that blockcopolymers form simple complexes with photosensitizing drugs. The typeof complexes formed was found to be codependent on the specific blockcopolymer and the specific photosensitizer utilized. These complexes maybe in forms such as micellar, emulsion, gel, matrix or transition phasesbetween the defined states.

[0079] Another observation of the invention is that certain copolymersin the poloxamer series spontaneously form micelles with thephotosensitizer drug. Micellar formulations have been produced in thelaboratory scale using the thin film method. For large scale drugproduction, the drug-copolymer and other components can be combinedusing techniques such as, but not limited to, spray or freeze drying, orthe Wurster-type coating process (Wurster, J. Amer. Pharm. Assoc.48:451, 1959) to form granules which will provide a higher surface areafor hydration or reconstitution. When forming micelles, it is preferredthat block copolymers of the above formula with a=60-80 and b=10 to 40units in length are used.

[0080] The invention also revealed that certain copolymers in thepoloxamer series spontaneously form a simple, stable bicomponent oil inwater (O/W) emulsions on simply hand-shaking with water or osmoticallybalanced aqueous solutions. The emulsion particle size in thesepreparations is small enough for intravenous administration (filterablethrough 0.2 (m filtration membranes), and particle size is retained over76 hours without any loss of drug on filtration. This, in conjunctionwith the knowledge that emulsions can be stabilized as reconstitutablesolid state preparations, makes the preparations highly viable asformulations for hydrophobic photosensitizing drugs.

[0081] Drugs could be incorporated directly into the block copolymer asdescribed in the Example section, or using minimal amounts of aninjectable solvent. Direct dissolution of photosensitizers inpoloxamers, particularly those in semi-solid or liquid form at ambientor body temperatures, would also provide useful ointments for topicaland mucosal applications. Alternatively, drug dissolved in minimalamounts of a non-toxic solvent may be added to an aqueous suspension ofthe block copolymer if it does not interfere with drug-copolymerinteractions, or destabilize the formulation in any other way.

[0082] Further, gel and matrix forming copolymers have been useful forcontrolled or sustained release, as well as delivery systems that can betriggered, and are prepared at higher polymer concentrations than thosedeemed suitable for parenteral formulations. Gelling of block copolymersat temperatures above ambient has been exploited in order to form ahigher viscosity drug release reservoir in contact with the lesion,either topically or onto mucosal area be treated. This allows arelatively non-invasive spraying of medicament onto affected areas, withgood contact maintained between the lesion to be treated and the drugformulation prior to light exposure.

[0083] The preferred block copolymers are those that can form stablecomplexes with a photosensitizer drug of interest. The more preferredcopolymers are the ones that form stable emulsions and/or micelles withthe photosensitizers, or undergo gel formation at body temperature.Other preferred copolymers are liquefied to permit a medicament, such asa photosensitizer, to be dissolved directly in the absence of a solvent.Poloxamers in liquid form act as highly effective solvents in whichhydrophobic drugs can be directly dissolved. Examples 2 and 3 belowillustrate this embodiment of the invention by demonstrating thatdifferent types of hydrophobic photosensitizers such as BPD-MA and B-B3can be dissolved in liquefied poloxamers.

[0084] Surprisingly, it appears that the nature of the drug can alsoinfluence the characteristics of the block copolymer in aqueoussolution. Block copolymers tested independently of the drug gave moreviscous solutions than in the presence of the drug substance. Withoutbeing bound by theory, the reason for this observation may be due toearlier induction or promotion of micelle formation by hydrophobicinteractions of the drug substance with the PPO block in the case ofpoloxamer. Depending on the nature of the active material, itsinteraction with the block copolymer might alter formulationcharacteristics e.g. serve to enhance formulation stability by promotingmicellization or altering emulsion characteristics. It is now generallyaccepted that certain block copolymers do, form micelles in aqueoussuspensions under certain conditions (Alexandridis et al. Macromolecules27:2414-2425, 1994).

[0085] For parenteral administration the most preferred block copolymersare those that form micelles with the photosensitive compound in theformulation. Water-soluble drugs might also benefit from the presence ofhydrophilic polymers to prevent chemical degradation, e.g. hydrolysis(Collett et al. J. Pharm. Pharmacol. 31 (suppl.) P80, 1979) during themanufacturing process, or storage, or improved ease of reconstitution inthe clinic.

[0086] More preferred for parenteral micellar formulations of highlyhydrophobic drugs are the family of poloxamers with the highestcommercially available molecular weight of PPO (n=60-80), and those with%PEO in the 20-40% range. For more water soluble formulations,non-micelle forming, hydrophilic polymers from the entire range could beutilized (PEO=40-90%). Emulsion forming polymers (%PEO=10-20%) might beuseful for certain hydrophobic and amphiphilic drugs. Poloxamers arenon-hygroscopic with water content of less than 0.5% w/w on exposure tothe atmosphere. Gel formation takes place in aqueous solutions in thehigher molecular weight polymers and is concentration and temperaturedependent. For instance, Pluronic® P123 gels at concentrations greaterthan 20% w/v at ambient temperature conditions. Gelling or viscosity isenhanced at body temperature, which could prove useful for prolongingcontact time of topical ocular and enteral formulations with the lesionsto be treated using PDT.

[0087] As an illustration of one embodiment of the invention, the blockcopolymer poloxamer series and in particular P123 has been extensivelyexamined. Therefore any poloxamers or block copolymer, in general, thathas similar characteristics, as P123 would be useful in this invention.Preferably, the block copolymers are effective in the concentrationrange of 0.005% to 20% w/v, more preferably in the range of 2 to 20% w/vfor parenteral formulations, and 0-100% for topical, enteral and ocularformulations. Poloxamers in liquid form act as highly effective solventsin which hydrophobic drugs can be directly dissolved. Poloxamers inliquid or paste form at ambient temperatures can be employed as liquidsor ointments for application.

[0088] P123 has been shown to be highly effective for formulating arange of tetrapyrrolic hydrophobic drug substances, such as the A, B, Cand D ring compounds. In the Example section below, formulation of thefollowing A-ring compounds: BPD-MA, A-EA6, A-B3; B-ring compounds;B-EA6, and B-B3; and other photosensitizers such as dihydroxychlorinsand pyropheophorbides, with P123 illustrate the versatility of thisparticular block copolymer. This includes A-ring compounds such asBPD-MA where block copolymers could be used to formulate an alternativeproduct to a concentration as high as 4 mg/ml in 10% P123, and alsoA-EA6 and A-B3, all of which formulate very readily. B-ring compoundshave lower drug loading characteristics, but concentrations ofapproximately 1.8 mg/ml are typical for B-B3, and lower for B-EA6. Awide range of other compounds e.g. pyropheophorbides and variousdihydroxychlorins also formulate with ease to give final formulations at2 mg/ml in 10% P123 in non-optimized systems. Therefore both the drugloading, and stability could be improved further by adjustments tocomposition, pH, and/or methodology of formulation. Surprisingly, withBPD-MA, greater drug loading was achieved in formulations with P123 thanwith any other tested poloxamer. This was also borne out with B-ringcompounds, which were the most stable in P123 than in any of the othertested poloxamers, under the given conditions.

[0089] Preferred poloxamers of the invention include poloxamer 403(P123), poloxamer 407 (P127), poloxamer 402 (P122), poloxamer 181 (L61),poloxamer 401 (L121), poloxamer 185 (P65), and poloxamer 338 (F108).

[0090] In another embodiment it is preferred that the molar ratio of thecopolymer to drug be equal to or greater than one. The present inventionincludes the discovery that increased ratios of copolymer to drugimproves drug “loading” into the disclosed medicament and carrier, ormedicament and carrier and solid support, formulations.

[0091] In one embodiment of the invention, blends of block copolymerswith other ionic and non-ionic surfactants, and other materials may beused to supplement, or compensate for physical and chemical propertieslacking in the primary copolymer. For instance, the “oiliness” ordifficult hydration of a certain copolymers may be counteracted byinclusion of one or more hydrophilic copolymer(s) or other surfactantfamilies such as, but not limited to PEG, PVP, Triton, Tween, oramphiphilic substances such as bile salts and lipids or lipidderivatives. As an illustration of this embodiment, blendingPluronic®127 and P123 is demonstrated in Example 15 below. This examplealso illustrates that blending poloxamers of different characteristicsimproves subsequent hydration and stabilizes the formulation compared tosingle poloxamer. Thus specific blends of block copolymers arecontemplated for use in the invention in combination with medicaments ingeneral, and photosensitizers in particular.

[0092] Mixed micelle systems have been shown to be highly effective indrug stabilization (Krishna et al. Journal 52, 6, 331-336, 1998).Micelles composed of hydrophobic drug-hydrophobic copolymer might bestabilized in aqueous suspension upon addition of one or morehydrophilic copolymer(s), or other surfactant families such as, but notlimited to PEG, PVP, Triton and Tween. Ionic surfactants could beenvisaged to embed themselves into the hydrophobic micelle with thehydratable headgroup providing high charge density at the micelle waterinterface. A similar effect might be achieved by blending blockcopolymers with a low molecular weight, highly water-soluble blockcopolymer or other surfactant material but not limited to bile salts andtheir derivatives, fatty acid derivatives, amino acids or other chargedhead groups. In another embodiment of the invention, photosensitizerscan be formulated in mixed micelle systems of ionic and non-ionicpolymers. Mixed micelles have been shown to effect drug stabilization(Chow & Bernard, J. Pharm Sci, 70, 8, 924-926, 1980, Krishna et al.Journal 52, 6, 331-336, 1998).

[0093] In yet another embodiment, photosensitizers can be formulated assimple oil in water (O/W) emulsions or W/O/W emulsions for formulationof photosensitizers using block copolymers. Certain poloxamers e.g.,Pluronic® L61, L121, L122 spontaneously form emulsions in the absence ofemulsifiers, or other stabilizing additives. Additionally, formulationsof L122 can be filtered through 0.2 μm sterilization filters with noloss of drug, and therefore suggesting a very small particle size. Theseemulsions have been found to be stable over several days (see Table 3below).

[0094] In an additional embodiment, hydrophobic copolymers with andwithout photosensitizers could be used as an adjunct to PDT, to improvethe therapeutic index of the PDT treatment in their capacity asimmunoadjuvants, e.g. in the treatment of metastatic lesions, dispersetumors or inflammatory lesions with microbial or autoimmune involvement.

[0095] In a further embodiment, the gelling properties of blockcopolymers can be utilized for preparing ocular formulations.Photosensitizing drugs can be formulated in block copolymer for eyedrops for ocular lesions to be treated; for example, hypervascularisedareas in macular degeneration, those induced by irritants e.g. excessiveexposure to UV. On account of the detergency and surfactant properties,intra-ocular formulations of photosensitizers in poloxamers (or post PDTwashes) would aid in clearing away of cellular debris generatedfollowing localized PDT e.g. for glaucoma and other conditions.

[0096] Moreover, topical and mucosal copolymer formulated preparationsare applicable, but not limited to, mucoadhesive preparations forinflammatory and autoimmune disorders for example, inflammatory boweldisease alopecia, psoriatic lesions.

[0097] In another embodiment the surfactant properties of copolymerformulations could be exploited to enhance dermal penetration ofphotosensitizing drugs, or that of psoriatic and other lesions.Penetration of the blood brain barrier by poloxamers has also beendocumented and could prove beneficial in the PDT treatment of braintumors or other disorders. (See Kabanov et al., J. ContrRel. 22,141-158, 1998).

[0098] In yet another embodiment, cellular uptake of photosensitizerscan be accelerated using copolymer formulations. The applicants haveshown in Example 11 below that cellular uptake of photosensitizers isaccelerated by utilizing poloxamer formulations.

[0099] In a further embodiment the copolymer formulations can be used toinduce the permeabilization of cellular membranes of thephotosensitizers. Cellular internalization of the drug and itsintracellular localization is critical in determining the final outcomeof PDT. The wetting capacity of copolymers to induce permeabilization ofcellular membranes could be exploited using compositions either with orwithout photosensitizers.

[0100] Parenteral administration of block copolymers would be useful intreating all the disorders mentioned above, particularly where treatmentor elimination of microvasculature is required. The advantage withpoloxamers is that it can be used to formulate highly hydrophobicphotosensitizer drugs. Poloxamers have been found to be useful in theinvention for formulation of hydrophobic photosensitizer drugs becauseof their high solubility in both aqueous systems and volatile solventsin which hydrophobic compounds such as BPD-derivatives display goodsolubility.

[0101] In another embodiment administration of block copolymerformulation of photosensitizers could be used for the treatment ofvarious types of cancers. Example 29 illustrates reduction of tumorrecurrence in tumor mice model, which were treated with poloxamerphotosensitizer formulation. In a further embodiment block copolymersallows both a greater proportion of the medicament to target tissuescompared to other formulations. This is illustrated in Example 29 wherepoloxamer formulations were compared to liposomal formulations using amice tumor models.

[0102] Preferably, solvents used in the invention when medicaments orphotosensitizers are not dissolved into a liquefied carrier, include anyorganic volatile solvent or mixture of solvents that are capable ofdissolving the carrier and photosensitizer. The choice of solvent to useis based in part on the hydrophobicity of photosensitizers and type ofcarriers, and the choice can be readily made, or made upon routineexperimentation, by the skilled artisan. Exemplary solvents used toillustrate this invention include, but are not limited to, methylenedichloride and ethanol.

[0103] The following processes can be used for formulations in theabsence, and even the presence, of a solid support. Depending on thestate of the medicament-carrier mixture and whether the medicament islabile, there are a number of ways of removing the fluids that may bepresent in the formulation mixture. Spray-drying techniques can be usedfor medicament-carrier that is in a liquid (molten or in solution)state. For block copolymers that revert to solid state on cooling, thespray dried product can be further micronized or ground to increase thesurface area for hydration. Wurster-type technology can be used forsemi-solid block copolymers to envelope or coat using exo-support, likea sugar, to prevent agglomeration of the spray dried particles.Supercritical fluid process is a single step process that can accomplishremoval of fluids (solvents) from a mixture and results in granules. Thegranules produced by this process are generally highly porous and resultin rapid hydration. This process can be used for medicament and carriermixture. Supercritical fluid using CO₂ has been used for preparingpolymeric microparticles and the advantages over other methods have beendiscussed by Bodmeier et al. (Pharm. Res. 12 (8): 1211-1218, 1995). Itis highly preferred that supercritical fluids be used for forminggranules for both liquid and solid block copolymers.

[0104] The solid product from the above processes can be subsequentlyhydrated or combined with alternative formulations depending on the modeof application or usage for instance, mixing with ointment bases fortopical applications.

[0105] Hydration of the medicament carrier with or without the supportmay be accomplished by addition of an aqueous based solution. The choiceof aqueous solution may depend on the components of the formulationmixture and how the hydrated complex is to be used. The aqueous basedsolution may be water or buffer, which may or may not contain variousexcipients or stabilizers. The hydrated complex can be processed furtherif required, or lyophilized or otherwise desiccated for storage. Theformulation may be prepared under Good Manufacturing Procedures (GMP).If the components are not sterile, the formulation may be sterilized byany known method in the art. These include heat, filter, radiation, andsterilization under conditions suitable for the medicament-carriermixture.

[0106] C. Solid Supports

[0107] The supports useful in the invention include both endo- andexo-supports that permit improved hydration in comparison tomedicament-carrier formulations prepared without such supports. The roleof the support is to maintain the precursor medicament and carrierformulation in a dry state prior to hydration and use. The support ispreferably chosen so that it does not dissolve in the carrier or solventused to dissolve the medicament. Endo-supports are defined as anysupport that can be used for depositing the medicament and carrier onthe surface of the support and that allows for hydration of themedicament and carrier in an aqueous based medium. An exo-support isdefined as any support that partially or wholly coats or encloses orencapsulates the medicament and carrier mixture.

[0108] In one embodiment the support that is suitable for this inventionare those that are non-toxic, biodegradable, not soluble in organicvolatile solvents or carriers used for dissolving the medicament(photosensitizer), suitable for deposition or encapsulation of themixture, and suitable for hydration of the deposited mixture in anaqueous based medium.

[0109] It is preferred that the endo- and exo-support are finely dividedand porous such that hydration of the deposited mixture is promoted dueto increased surface area.

[0110] In one embodiment the endo-support material is soluble uponhydration of the deposited medicament (or photosensitizer) and carriermixture. Preferred endo-support material include, but are not limitedto, ionic salts, lactose, dextrose, sucrose, trehalose, sorbitol,mannitol, xylitol or a naturally occurring polymers and amino acids orderivatives thereof. The more preferred material is lactose and the mostpreferred is trehalose, which may function both as a solid support and ahydration aid for a medicament/carrier mixture. These embodiments areillustrated in Examples 16 to 20 below, which show the use of suchendo-supports for depositing formulations of photosensitizer and one orblend of block copolymer carriers. For illustration purposes thephotosensitizers tested were the A and B-ring tetrapyrroles, thecarriers were non-blended and blended block copolymers from thepoloxamer group and the endo-support were the hydratable sugars such aslactose or trehalose.

[0111] Blended poloxamers with dissolvable solid-supports were found tohydrate faster than blended poloxamers without the solid-support.Examples 17 to below demonstrate the use of blended poloxamers P123 andF127 with hydratable solid-supports lactose or trehalose.

[0112] In another embodiment the solid-support can be of material thatis insoluble in liquefied carrier, solvent, or aqueous based solutionbut allows for hydration of the deposited mixture from the surface ofthe solid-support. In the latter case the solid-support material ispreferably non-toxic, biodegradable and/or easily removed from thehydrated formulation. Such materials include any be any polymericmaterial that has been found to be suitable for therapeutic use orimplants.

[0113] As stated above, solvent removal may be by any process that doesnot damage the drug and removes the solvent from the blockcopolymer-photosensitizer drug mixture deposited on the solid-support.Examples of such processes include, but are not limited to, heat drying,microfluidization, spray-drying, Wurster technology, lyophilization, andthe use of super critical fluid granulation.

[0114] Additional discussion of solid supports for photosensitizerformulations is provided in the simultaneously filed U.S. patentapplication entitled “Supports for Photosensitizer Formulations” (Atty.docket no. 273012011700) which is hereby incorporated by reference inits entirety, as if fully set forth.

[0115] D. Pharmaceutical Compositions and Administration

[0116] The photosensitizer is formulated into a pharmaceuticalcomposition by mixing the medicament (or photosensitizing agent) withone or more physiologically acceptable carriers, i.e., carriers that arenon-toxic to recipients at the dosages, concentrations and modes ofadministrations employed. The medicament may be used in its solid formor dissolved in an appropriate solvent for addition to the carrier(solid or liquefied) or dissolved in an appropriate solvent. Preferredmixtures should be in appropriate solvents for dissolving bothmedicament and carrier, and at the desired degree of medicament purity.It is preferred that upon hydration, at the appropriate pH for themedicament, the photosensitizer and carrier form a complex whichfacilitates delivery of the photosensitizer to the target. Otheradditives and pharmaceutical excipients can also be added, during orafter formulation, to improve the ease of formulation, formulationstability, speed of reconstitution, delivery of the formulation. Theseinclude, but are not limited to, penetration enhancers, targeting aids,anti-oxidants, preservatives, buffers, stabilizers, solid supportmaterials. The composition may include osmoregulators if required, suchas but not limited to, physiologically buffered saline (PBS),carbohydrate solution such as lactose, trehalose, higherpolysaccharides, or other injectable material. A wide variety ofexcipients and stabilizers are known in the art and their use willdepend on the formulation type and application requirements. Thefunction of stabilizers is to provide increased storage stability incases where the photosensitizer or carriers are labile to heat, cold,light or oxidants or other physical or chemical agents. Other purposefor stabilizer may be for maintaining photosensitizer and/or carrier ina form appropriate for transport to and uptake at the target site.Depending on the solubility, the excipients or stabilizers may be addedeither prior to deposition step or after the hydration step.

[0117] The formulations of the invention may be incorporated intoconvenient dosage forms, such as capsules, impregnated wafers,ointments, lotions, inhalers, nebulizers, tablets, or injectablepreparations. Preferably, the formulations of the invention areadministered systemically, e.g., by injection. When used, injection maybe by any known route, preferably intravenous, subcutaneous,intradermal, intramuscular, intracranial or intraperitoneal. Injectablescan be prepared in conventional forms, either as solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Intravenous preparations can beadministered as a bolus injection or by controlled infusion followingprior dilution if deemed necessary. Controlled intravenous injection isespecially preferred following reconstitution, or dilution of thereconstituted drug substance in a physiologically acceptable aqueouspreparation.

[0118] Solid or liquid pharmaceutically acceptable carriers may beemployed. Solid carriers include starch, lactose, calcium sulfatedihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,magnesium stearate and stearic acid. Liquid excipients include syrup,peanut oil, olive oil, saline, water, dextrose, glycerol and the like.Similarly, the carrier or diluent may include any prolonged releasematerial. When a liquid carrier is used, the preparation may be in theform of a syrup, elixir, emulsion, soft gelatin capsule, sterileinjectable liquid (e.g., a solution), such as an ampoule, or an aqueousor nonaqueous liquid suspension. A summary of such pharmaceuticalcompositions may be found, for example, in Remington's PharmaceuticalSciences, Mack Publishing Company, Easton Pa. (Gennaro 18th ed. 1990).

[0119] The pharmaceutical preparations are made following conventionaltechniques of pharmaceutical chemistry involving such steps as mixing,granulating and compressing, when necessary for tablet forms, or mixing,filling and dissolving the ingredients, as appropriate, to give thedesired products for oral or parenteral delivery, including topical,transdermal, mucosal, intravaginal, intranasal, intrabronchial,intracranial, intraocular, intra-aural and rectal administration. Thepharmaceutical preparations may also contain minor amounts of nontoxicauxiliary substances such as wetting or emulsifying agents, pH bufferingagents and so forth. Pharmaceutical compositions formulated for timedrelease may also be prepared. The preparations may includeosmoregulators if required, such as but not limited to, physiologicallybuffered saline (PBS), carbohydrate solution such as lactose, trehalose,higher polysaccharides, or other injectable material.

[0120] For topical application, the compound may be incorporated intotopically applied vehicles such as a salve or ointment. The carrier forthe active ingredient may be either in sprayable or nonsprayable form.Non-sprayable forms can be semi-solid or solid forms comprising acarrier indigenous to topical application and having a dynamic viscositypreferably greater than that of water. Suitable preparations include,but are not limited to, solution, gels, suspensions, emulsions, creams,ointments, powders, liniments, salves, eye drops, and the like. Ifdesired, these may be sterilized or mixed with auxiliary agents, e.g.,preservatives, stabilizers, wetting agents, buffers, penetrationenhancers, or salts for influencing osmotic pressure and the like.Preferred vehicles for non-sprayable topical preparations includeointment bases, e.g., polyethylene glycol-1000 (PEG-1000); conventionalcreams such as HEB cream; gels; as well as petroleum jelly and the like.

[0121] Also suitable for topic application are sprayable aerosolpreparations wherein the compound, preferably in combination with asolid or liquid inert carrier material, is packaged in a squeeze bottleor in admixture with a pressurized volatile, normally gaseouspropellant. The aerosol preparations can contain solvents, buffers,surfactants, preservatives, and/or antioxidants in addition to thecompounds of the invention.

[0122] For the preferred topical applications, especially for humans, itis preferred to administer an effective amount of the formulation to atarget area, e.g., skin surface, mucous membrane, eyes, etc. This amountwill generally range from about 0.001 mg to about 1 g per application,depending upon the area to be treated, the severity of the symptoms, andthe nature of the topical vehicle employed.

[0123] The formulations of the invention may be given in combinationwith one or more additional compounds that are used to treat the diseaseor condition. For treating cancer, the formulations are given incombination with anti-tumor agents, such as mitotic inhibitors, e.g.,vinblastine; alkylating agents, e.g., cyclophosphamide; folateinhibitors, e.g., methotrexate, pritrexim or trimetrexate;antimetabolites, e.g., 5-fluorouracil and cytosine arabinoside;intercalating antibiotics, e.g., adriamycin and bleomycin; enzymes orenzyme inhibitors, e.g., asparaginase; topoisomerase inhibitors, e.g.,etoposide; or biological response modifiers, e.g., interferon. In fact,pharmaceutical preparations comprising any known cancer therapeutic incombination with the formulations disclosed herein are within the scopeof this invention.

[0124] The pharmaceutical preparations of the invention may alsocomprise one or more other medicaments such as anti-infectives includingantibacterial, anti-fungal, anti-parasitic, anti-viral, andanti-coccidial agents.

[0125] Typical single dosages of the formulations of this invention arebetween about 1 ng and about 10 g/kg body weight. The dose is preferablybetween about 0.01 mg and about 1 g/kg body wt. and, most preferably,between about 0.1 mg and about 100 mg/kg body wt. For topicaladministration, dosages in the range of about 0.01-20% concentration ofthe compound, preferably 1-5%, are suggested. A total daily dosage inthe range of about 1-500 mg is preferred for oral administration. Theforegoing ranges are, however, suggestive, as the number of variables inregard to an individual treatment regime is large, and considerableexcursions from these recommended values are expected.

[0126] Effective amounts or doses of the compound for treating a diseaseor condition can be determined using recognized in vitro systems or invivo animal models for the particular disease or condition. In the caseof cancer, many art-recognized models are known and are representativeof a broad spectrum of human tumors. The compounds may be tested forinhibition of tumor cell growth in culture using standard assays withany of a multitude of tumor cell lines of human or nonhuman animalorigin. Many of these approaches, including animal models, are describedin detail in Geran, R. I. et al., “Protocols for Screening ChemicalAgents and Natural Products Against Animal Tumors and Other BiologicalSystems (Third Edition)”, Canc. Chemother. Reports, Part 3, 3:1-112.

[0127] E. Storage & Handling

[0128] Poloxamer based formulations do not require extensive precautions(shielding from light and exposure to air (oxygen and moisture) as isoften recommended with phospholipids for liposomal formulations,especially in conjunction with photosensitizers. Thus thephotosensitizer drug substance would be the only labile material in thepoloxamer based formulations, compared to the conventional emulsion orliposomal based formulations, where in addition to the photosensitizer,phospholipids and other labile components would be present.

[0129] Poloxamer block copolymer formulations have an extended shelflife because poloxamers are chemically inert molecules with none of thehydrolytic and oxidative/photo-oxidative degradation problems,associated for instance with liposomal systems. Peroxide generation inunsaturated phospholipid systems contributes to propagation of freeradical processes, which can potentially degrade not only the lipidsthemselves, but also the active drug. Free radicals are not expected tobe generated in poloxamer systems to the same extent, and the need foradditives e.g. anti-oxidants, would be greatly reduced when compared tounsaturated liposomal formulations. Since poloxamers are synthetic,there is also no concern about potential transmission of biohazardousdisease vectors associated with animal derived products.

[0130] Poloxamer based formulation can be rapidly and easily bedeveloped for highly economic large scale manufacturing procedures. Dueto the simplicity and non-fragile nature of the components,manufacturing can be carried out in a single step prior to packaging forreconstitution by spray drying, lyophilizing or low heat drying from avolatile solvent, under conditions for Good Manufacturing Practices(GMP) conditions).

[0131] F. Drug Release

[0132] In liposomal formulations of BPD-MA, drug fluorescence isconcentration quenched due to its location in the liposomal membrane.This allows its release to plasma proteins to be monitored. This is notthe case for copolymer formulations lacking fluorescence quenching, inwhich case it is assumed that the drug is encompassed in itsnon-aggregated form in a more dynamic micellar system. It is thereforelikely to be released instantaneously in the presence of alternativedrug-binding molecules (such as lipoproteins) upon injection into thecirculation. Example 11 below shows the high level of association ofB-ring drugs with the lipoprotein fraction following a very briefexposure to human plasma.

[0133] G. Photodynamic Therapy

[0134] Preferably, electromagnetic radiation, such as from ultravioletto visible and infra red light, is delivered after administration of thecompositions and formulations of the invention. Also preferred in theinvention is the use of low-dose PDT. By “low-dose PDT”, it is meant atotal photodynamic therapy experience at substantially lower levels ofintensity than that ordinarily employed. Generally, there are threesignificant variables—the concentration of the photosensitizing drug,the intensity of the radiation employed and the time of exposure tolight, which determines the total amount of energy ultimately deliveredto the target tissue. Generally, an increase in one of these factorspermits a decrease in the others.

[0135] For example, if it is desired to irradiate only for a shortperiod of time the energy of irradiation or the concentration of thedrug may be increased. Conversely, if longer time periods of irradiationare permitted, lower irradiation intensities and lower drugconcentrations are desirable. In some instances, the combination of 0.15mg BPD-MA as a drug concentration and approximately 1 J/cm2 totalradiation from an appropriate radiation source provided successfulresults. The use of low dose PDT offers an additional advantage in theform of reducing the likelihood of PDT side effects such as damage tounintended tissues.

[0136] It is understood that the manipulation of these parameters willvary according to the nature of the tissue being treated and the natureof the photosensitizer (PS) employed. However, in general, low-dose PDTemploys combinations of the drug concentration, radiation intensity, andtotal energy values which are several fold lower than thoseconventionally used for destroying target tissues such as tumors andunwanted neovascularization. One measure might be the product of PSconcentration (e.g., in ng/ml)×intensity (e.g., in mW/cm2)×time (e.g.,in seconds). However, it is difficult to set absolute numbers for thisproduct since there are constraints on each of the parametersindividually. For example, if the intensity is too low, the PS will notbe activated consistently; if the intensity is too high, hyperthermicand other damaging effects may occur. Additionally, in some instances,ambient or environmental light available at the target cell or tissueundergoing PDT may be sufficient in the absence of additional deliberateirradiation.

[0137] Similarly, PS concentrations cannot vary over any arbitraryrange. There may also be constraints on the time during which radiationcan be administered. Accordingly, the product of the foregoing equationis only a rough measure. However, this approach may provide a convenientindex that can be adjusted according to the relative potency of the PSemployed, and in general, an increase in intensity would permit adecrease in time of irradiation, and so forth.

[0138] Having now generally described the invention, the same will bemore readily understood through reference to the following exampleswhich are provided by way of illustration, and are not intended to belimiting of the present invention, unless specified.

EXAMPLES

[0139] The following general comments on materials apply to thefollowing examples, unless otherwise noted.

[0140] BPD-MA, BPD derivative EA6, and B3 A and B ring compounds weresynthesized as described in the patents recited above. BPD-MA, A-EA6,B-EA6, A-B3, and B-B3 were obtained from QLT PhotoTherpeutics Inc.(Vancouver, B.C., Canada; QLT).

Example 1 Prescreening of Block Copolymers for Photosensitizer DrugLoading

[0141] The following example illustrates the pre-screening of blockcopolymers for utility in drug loading for intravenous delivery bystudying the aqueous suspension characteristics.

[0142] Although certain block copolymers have been used previously asemulsion stabilizers in various pharmaceutical formulations, blockcopolymers which themselves emulsify in aqueous suspension have not beenstudied in great detail for parenteral formulations. This has been dueto the greater difficulty in controlling and maintaining particle sizeduring manufacture and storage. Ideally, a stable micellar suspension ispreferred. For extended shelf life, the final formulation is required tobe in a dry form which is easily reconstituted for injection. Anacceptable minimum reconstituted drug concentration for an intravenousformulation is in the range of 1-2 mg/ml with at least 4 h postreconstitution stability in aqueous suspension. Important considerationsfor intravenous formulation are (i) delivery of drug in a non-aggregatedform, (ii) low viscosity preparations (iii) non-frothy preparations, and(iv) sterile filterability prior to drying. A criterion for hydrophobicdrug formulation is effective delivery to the plasma lipoproteins, whichact as intermediate drug carrier in vivo to tissues displaying highlevels of LDL receptors. These include hyperplastic tissues and thoseundergoing repairs, e.g. under inflammatory conditions.

[0143] In this experiment the copolymers were pre-screened for theirpotential as injectable drug formulation agents, starting with theexamination of their aqueous suspension characteristics at variousconcentrations i.e. whether they formed emulsions or solutions in water.The Pluronic® copolymers used in this and subsequent experiments wereobtained from BASF Corp. and are described in the following table withtheir PPO/PEO contents and molecular weights.

[0144] 5 ml suspensions of each Pluronic were made at 5%, 10%, 15% and20% w/v in physiologically buffered saline (PBS), pH 7.4. This wasfacilitated by sonicating the suspensions in a water bath (Aquasonic,250D, VWR Scientific) at 55° C. The suspensions were then examined andthe viscosity of each suspension was determined visually by thethickness of film left on vial wall as it was tilted, and by relativeease of filtration through 0.2 μm filters (Sterile Acrodisc 13, GelmanSciences). TABLE 1 PEO⁷ PPO⁸ MW Poloxamer¹ Pluronic ®² (a) (b) (g/mol)401 L³121  6 67 4400 402 L122 13 67 5000 403 P⁴123 21 67 5750 407 F⁵127⁶98 67 12000  338 F108⁶ 128  54 15000  181 L61  3 30 2000 185 P65 19 303400 188 F68⁶ 75 30 8350 124 L44⁶ 11 21 2200

[0145] Table 2 summarizes the qualitative results of the solutionappearance, viscosity and filterability of 5 to 20% weight by volume(w/v) concentration range of the different types of poloxamers in PBS.Generally, viscosity in both solutions and emulsions increased withPluronic concentration. Copolymers forming highly viscous suspensions(e.g. preparations at higher Pluronic concentrations) or those forminghighly unstable emulsions e.g. L61 were not further tested. Copolymerswith a lower PEO content less that 30% (L61, L121, L122) displayedlimited water solubility, and tended to form oily emulsions rather thanclear solutions. Under the above conditions, Copolymers that formedsolutions were those with a higher PEO content such as P123, P127, F68,F_(108,) and were tested further for drug loading at lowerconcentrations. TABLE 2 Solubility, viscosity and filterabilitycharacteristics of poloxamers 5% w/v 10% w/v 15% w/v 20% w/v Appearance/Appearance/ Appearance/ Appearance/ Pluronic Viscosity FilteredViscosity Filtered Viscosity Filtered Viscosity Filtered L121 Opaque YesOpaque Yes Opaque Yes Gels No emulsion emulsion emulsion L122 Frothy YesFrothy Yes Viscous Yes Gels No emulsion emulsion frothy emulsion P123Clear frothy Yes Clear frothy Yes Clear frothy No Clear frothy Nosolution solution/ solution/ solution/ slight medium medium viscosityviscous viscous F127 Clear Yes Clear Yes Clear Yes Clear No solution/lowsolution/ solution/ solution/ viscosity medium high high viscosityviscosity viscosity L61 Oily No Oily No Oily No Unstable No emulsion/emulsion/ emulsion/ emulsion low low low viscosity viscosity viscosityP65 Frothy Yes Frothy Yes frothy Yes frothy Yes solution/lowsolution/low solution/low solution/low viscosity viscosity viscosityviscosity F68 Clear Yes Clear Yes Clear Yes Slightly Yes solutionsolution solution viscous F108 Clear Yes Frothy Yes High No High Nosolution viscous viscosity viscosity solution solution solution

Example 2 Photosensitizer Drug Loading of BPD-MA Using PEO-PPO-PEO BlockCopolymers

[0146] The following example illustrates the utility of block copolymersfor drug loading of an A-ring tetrapyrrolic compound.

[0147] In this experiment the use of copolymers for drug loadingcapability and formulation stability over a 3 day period was examinedusing the photosensitizer drug BPD-MA. The criteria for choosing thecopolymers were based on the solution and viscosity characteristicsdescribed in Example 1. The ‘melt’ method is used for the preparationand screening of the large number of samples and is described asfollows. At temperatures above 50° C., poloxamers are in their moltenstate and serve as excellent solvents for tetrapyrrolic compounds, thusavoiding the need for pre-dissolution of drugs in organic solvents. 5 mgof BPD-MA was dissolved with the aid of vortex mixing and sonication at55° C. into the polymer ‘melts’ to give a final concentration of 5% to20% w/v of the respective Pluronic. To each melt sample, 2.5 ml of PBSwas added to give a final BPD-MA concentration of 2 mg/ml. Samples wereallowed to equilibrate to room temperature before drug loading wasdetermined at time zero (To). 1 ml of suspension was removed forcentrifugation (Microfuge, 14,000 rpm, 30 min), and the rest filteredthrough 0.2 μm filters (Millipore). The filtrate was diluted 1:100 inPBS and the absorbance at 690±3 nm determined (uv-vis spectrophotometerBeckman DU-6401). This procedure was repeated 72 hours later followingstorage at room temperature and the absorbance measurement (T₇₂).

[0148] The following table summarizes the results of the aboveexperiment. TABLE 3 Absorbance (A₆₉₃) of BPD-MA of filtered (F) andcentrifuged (C) samples after hydration. 5% w/v 10% w/v 15% w/v 20% w/vPluronic C/F¹ T₀ T₇₂ T₀ T₇₂ T₀ T₇₂ T₀ T₇₂ L122 C 0.51 0.64 0.74 0.76 0.80.41 N/D² N/D F 0.61 0.53 0.76 0.73 0.43 0.57 N/D N/D P123 C 0.44 0.620.86 0.66 N/D N/D N/D N/D F 0.64 0.66 0.69 0.58 N/D N/D N/D N/D F127 C0.74 0.64 0.67 0.67 0.81 0.88 N/D N/D F 0.62 0.63 0.66 0.64 0.87 0.83N/D N/D P65 C 0.1 0.02 0.43 0.36 0.9 0.73 1.0 0.97 F 0.02 0.09 0.35 0.430.81 0.78 0.97 0.97 F68 C 0.3 0.25 0.13 0.05 0.09 0.06 0.3 0.07 F 0.250.33 0.06 0.13 0.11 0.07 −?? 0.24 F108 C 0.17 0.19 0.72 0.65 N/D N/D N/DN/D F 0.59 0.58 0.68 0.73 N/D N/D N/D N/D

[0149] The results show that highest drug loading using 5% w/vcopolymers gave A693 ranging from 0.5 to 0.7 for L122, P123 and F127 inboth centrifuged and filtered preparations. These copolymers have thehighest PPO content (67 Units). Drug loading using 10% w/v copolymershowed highest drug loading with L122, P123 and F127 and F108 (PPO 54units) with A693 ranging from 0.58 to 0.76. P65 (PPO 30 units, PEO 19units) showed minimal incorporation at 5 and 10% w/v but totalincorporation at 15 and 20% w/v. Drug loading was greater than in F68that has the same number of PPO units. Solution forming poloxamers suchas P123, L122 and F127, show little discrepancy between centrifuged andfiltered samples, suggesting that both procedures were equally effectivein removing unincorporated photosensitizer drug aggregates from theformulations. The A₆₉₀ reading were comparable between day 0 and day 3which implied that there was no loss of stability of BPD-MA formulationsin Pluronic following 3 days storage.

[0150] Based on the observation that greater drug loading is dependenton lower water solubility (low PEO) within a given PPO group, butwithout being bound by theory, it seems possible that micelle formationis important for stabilization of highly hydrophobic drug substances. Areason why F68 does not perform well may be because of its high watersolubility. The extended PEO chains (PEO 75 units) would not beconducive to micelle formation.

Example 3 Photosensitizer Drug Loading of B-B3 Using Pluronic BlockCopolymers

[0151] The following example illustrates the utility of block copolymersfor drug loading of B-ring tetrapyrrolic compounds, and maintaining thedrug in a non-aggregated form.

[0152] For this experiment copolymers were examined for drug loadingcapability and formulation stability over a 24 h period using the drugB-B3. The experimental procedure is the same as described in Example 2with the following exceptions. The copolymers were tested at 10%, 15%and 20% w/v. For convenience centrifugation rather than filtration wasused to eliminate unincorporated drug prior to absorbance measurement.It has previously been observed that aggregates of B-ring compounds havea characteristic red shifted, high extinction absorbance at 730 nm±10nm, which takes place at the expense of the typical 690 nm absorbanceattributed to monomers. The 730 peak correlates with sub-optimalformulation conditions, and has proved useful for evaluation offormulation quality. Dissolution of green crystalline B-ring compoundsin -melted poloxamers resulted in a reddish brown solution absorbingentirely at 690 nm. Similar color was observed in stable formulations ofB-ring compounds in aqueous suspensions of poloxamers.

[0153] Table 4 shows results of B-B3 drug loading using various blockcopolymers. Overall, the results for B3-B drug loading displayed thesame general pattern as for BPD-MA as seen in Example 2, but with lowerdrug loading. Polymers L122, P123 and F127 showed the highest drugloading. Unlike loading of BPD-MA in P65 (Example 2), the drug loadingwas comparable to the PPO 67 unit group, this was not the case for B-B3,even at the highest P65 concentrations tested. TABLE 4 Absorbance(A_(693nm)) of B-B3 formulation following hydration and centrifugation10% w/v 15% w/v 20% w/v Pluronic ® T₀ T₂₄ T₀ T₂₄ T₀ T₂₄ L122 0.54 0.500.5 0.56 N/D N/D P123 0.52 0.53 N/D N/D N/D N/D F127 0.57 0.4 0.51 0.48N/D N/D F108 0.1 0.015 N/D N/D N/D N/D P65 0.07 0.07 0.15 0.13 0.36 0.24F68 0.03 0.025 0.02 0.02 0.03 0.03

[0154] On 1:100 dilution, the P123 formulation displays a 690 nmabsorbance in PBS which is similar to that in organic solvents e.g.methanol suggesting a similarly hydrophobic environment for the drug inthe Pluronic formulation. Twenty minutes following dilution produced a730 nm peak in the F127 formulation (results not shown), but not in the10% w/v P123 or L122 formulations. This is again indicative of amicellar organization for the poloxamers in aqueous suspensions,particularly in those with an intermediate PEO content >10% w/w. Highlywater soluble polymers such as F127, form unstable preparationsparticularly on dilution, as the ratio F127:drug decreases resulting inmicelle destabilization with consequent drug aggregation.

[0155] Centrifugation of unstable formulations (P65, F68, F108) resultedin an aggregated drug pellet absorbing predominantly at 730 nmwavelength, even on suspension in 100% fetal bovine serum. This confirmsthat the 730 nm peak may indicate low non-aggregated drugbioavailability to plasma lipoproteins and therefore should be avoidedin formulation of B-ring compounds.

Example 4 Drug Loading of B-EA6 and B-B3 Using Block Copolymers and ThinFilm Approach

[0156] The following example describes an alternative method for B-ringhydrophobic drugs (B-B3 and B-EA6) that were previously described asbeing difficult to formulate, and to do so using smaller quantities ofdrug and block copolymers. Although the melt method described in Example2 works well for formulating hydrophobic drugs, it requires constantstirring and vortex mixing to maintain the drug in contact with thesmall volume of block copolymers used. The smallest volume that could beprepared using such a method was approximately 5 ml. Creating a thinfilm from a solution of both the drug and Pluronic in a volatile organicsolvent on the other hand, allows a larger surface area for fasterhydration.

[0157] The B-ring drugs B-EA6 and B-B3 were tested by the followingformulation method. 5 mg of the drug and 0.5 g Pluronic were dissolvedin methylene chloride (CH₂Cl₂) and combined to give final volume of 2.5ml in a round bottom flask. The solvent was removed by rotaryevacuation, and the resultant thin film hydrated with 2.5 ml PBS at 50°C. in a sonication bath. After cooling to room temperature (1-2 hours),samples were centrifuged to remove unincorporated drug, and A₆₉₀ of1:100 dilutions was determined.

[0158] The results of formulating B-B3 and B-EA6 by the poloxamer basedthin film approach are summarized in Table 5. TABLE 5 Absorbance(A₆₉₀)Pluronic (10%) B-B3 B-EA6 P123 0.8 0.315 L122 0.6 0.275 F127 0.4 0.08

[0159] It was surprising to note that B-EA6 could be formulated withblock copolymers because of earlier poor results obtained with othercarriers and liposomal formulation attempts. B-B3 was more readilyformulated in poloxamers compared to B-EA6 under the above conditions.The order of formulation efficiency remained the same as observed inExample 3, i.e. F123>L122>F127. Both drug preparations in 10% F127developed the 730 absorbance peak within 15 min of dilution in PBS. Thiswas indicative of formulation destabilization and drug aggregation inaqueous suspensions, perhaps due to an unstable micellar structure.

Example 5 Hydrophobic Photosensitizer Drug Loading Using BlockCopolymers

[0160] The following example illustrates one embodiment for hydrophobicdrug loading using block copolymers.

[0161] Unless otherwise stated, the following protocol was used for allsubsequent formulation of the photosensitizer drugs in poloxamers:

[0162] 1 to 2 mg drug and 25-100 mg Pluronic are combined in methylenedichloride (CH₂Cl₂) to yield drug concentration of 1 mg/ml. CH₂Cl₂ isremoved rapidly by rotary evacuation (Rotavapor R-124, Bucchi B172Vacobox pump) at 50°, at maximum speed of rotation. Once a steadyminimum pressure is achieved, the flask is held under vacuum for afurther 20-30 min. The resulting thin film is hydrated with 1 ml ofphysiologically buffered saline (PBS, pH 7.4) or 9.5% w/v lactose, usinghand swirling (with glass beads) at 23° C., to give a final drugconcentration of 1 or 2 mg/ml, 2.5-10% (w/v) Pluronic as required.Samples are kept overnight at room temperature to allow unincorporateddrug to fall out, and then spun at 14,000 rpm {Eppendorff, Microfuge}for 30 min. Supernatant is decanted off into a fresh Eppendorff vial,and diluted 1:100 in the iso-osmolar medium used for thin film hydration(PBS or lactose) for determination of absorbance 690 nm (A₆₉₀).Formulations are stored at 4° C. or frozen at −20° C. for long termstorage.

Example 6 Protocol for Liposomal Photosensitizer Drug Formulation

[0163] The following example describes a protocol for liposomalpreparation of hydrophobic photosensitizers. It is based on existingmethodology (Hope et al., Biochim. Biophys. Acta 812, 55-65, 1985).

[0164] 5 mg drug and lipids (40% EPG in DMPC) are combined in CH₂Cl₂ ata drug to lipid ratio of 1:10 w/w in 250 ml round bottom flask. Themaximum concentration of drug in solvent is 2 mg/ml. CH₂Cl₂ is removedrapidly as described in Example 5. The resulting thin film is hydratedwith 2.5 ml lactose solution (9.5% w/v) using hand swirling with glassbeads at 40° C. Extrusion using Model 4T (Lipex Biomembranes Inc. B.C.,Canada) is carried out with the thermostat set at 40° C. Themultilamellar vesicles (MLVs) arising from hydration steps of theliposomal formulation were also examined under the microscope. MLVs aresuccessively extruded 5 times through each of the 400 nm, 200 nm and 100nm polycarbonate membranes (Nuclepore PC, Costar). Extruded samples werediluted 1:100 in PBS (pH 7.4) and the absorbance determined at 690 nmwavelength.

Example 7 Comparison of Liposomal and Block Copolymer PhotosensitizerFormulations

[0165] This example demonstrates that micellar formulations ofphotosensitizers using block copolymers were either comparable orsuperior to the liposomal formulations.

[0166] In this experiment liposomal and block copolymer (micellar)photosensitizer formulations of A- and B-ring compounds of EA6 and B3were compared. Each of the photosensitizer samples was prepared at afinal drug concentration of 2 mg/ml. The block copolymer P123, and theliposomal formulations were prepared as described in the Examples 5 and6, respectively.

[0167] Table 6 shows the results of the photosensitizer drug loadingusing 10% P123 and liposomes. The A-rings could be formulated usingliposomes but formulation of the B-ring compounds was not veryefficient. P123 was found not only able to formulate the A-ringcompounds but also the B-ring compounds. With the exception of A-B3, theoverall results for the drug loading showed that the P123 formulationswere either superior or comparable to the liposomal formulation. TABLE 6Liposome P123¹ Drug mg/ml mg/ml  A-EA6 0.98 1.82 A-B3 1.84 1.33 B-EA0.06 0.37 B-B3 Very low 1.24

[0168] It was observed that in the liposomal formulation the step ofhydration of thin film of the A-ring compounds took place readily withthe total drug incorporation into MLVs. Microscopic examination did notreveal presence of aggregates. Extrusion took place readily under lowpressure without significant loss of drug. In contrast, MLVs arisingfrom hydration of B-ring films were unevenly shaped, with drugaggregates and crystalline structures commonly present. These crystalswere problematic because they caused filter blockage during theextrusion process and resulted in significant drug loss. Liposomalformulation with B-ring preparations resulted in very small quantitybeing incorporated in the liposomes (Table 7).

[0169] Formulation with block copolymer P123 resulted in ready hydrationof thin films of the A-ring compounds. For the B-ring compounds therewas greater drug incorporation using P123 compared to the liposomalformulation.

[0170] The above example demonstrates that block copolymer P123 readilyincorporated different types of photosensitizers with either similar orsuperior drug loading compared to the liposomal formulations.

Example 8 Formulation of Dihydroxychlorins in Block Copolymers

[0171] The following example illustrates the use of block copolymer forformulating dihydroxychlorin photosensitizers.

[0172] In this experiment the following three selected dihydroxychlorinswere examined for formulation with 10% P123. Each of the drugs wasprepared to a final concentration level of 1 mg/ml and the formulationprotocol used is described in Example 5. These compounds were preparedas described in U.S. patent application Ser. Nos. 09/551,159 and09/551,160, both filed Apr. 14, 2000, and No. 60/129,324, filed Apr. 14,1999, all three of which are hereby incorporated by reference as iffully set forth. One of these compounds, JM 4, was further tested fordrug incorporation using 2.5 to 10% P123. TABLE 7 ID No. Formula JM3T(m-OH)PC = 5,10,15,20-tetra (meta-hydroxyphenyl)-2-3-dihydroxychlorinJM4 T (p-Me) PC = 5, 10, 15, 20-tetra (para-methylphenyl)-2,-3-dihydroxychlorin JM24 H₂DPC(OH)₂

[0173] All of the above dihydroxychlorin compounds were formulated withease using 10% P123. The compounds underwent total incorporation with nopellet formation on centrifugation either directly following formulationor 24 h later. The micelle size ranged from 15 to 20 nm measured bylaser light scattering (Submicron Particle Sizer Model 370, NICOMP,Santa Barbara, Calif.). The formulation was also found to be stablefollowing overnight storage.

[0174] Table 8 shows the results of drug incorporation using differentconcentration of the copolymer P123. The readings following overnightstorage and centrifugation. Formulation of JM4 at 2 mg/ml showed thatthe amount of drug incorporated was found to be dependent on theconcentration of polymer in the formulation. TABLE 8 Incorporation P123% w/v mg/ml 2.5 0.92 5 1.43 10 2.00

[0175] The above example demonstrates the versatility of the P123 blockcopolymer for formulating different types of photosensitizers.Additionally this example shows that the concentration of the blockcopolymer will dictate the level of photosensitizer incorporation.

Example 9 Plasma Distribution of Photosensitizers Delivered by BlockCopolymer and Liposomal Formulations

[0176] This example illustrates that B-ring photosensitizers formulatedwith the block copolymer L123 are delivered with the same or greaterefficiency to the lipoprotein fraction of the plasma compared to thestandard liposomal formulation of an A-ring compound, BPD-MA.

[0177] In this experiment liposomal, block copolymer and dimethylsulfoxide (DMSO) formulations of the B-ring compounds, B-EA6 and B-B3were examined for their partitioning between the different components ofhuman plasma. BPD-MA liposomal formulation was used as the standard andthe DMSO as a control. Pluronic micellar and liposomal formulations ofthe photosensitizers were prepared as described in Examples 5 and 6,respectively. DMSO formulation was prepared by direct dissolution of thedrug in DMSO.

[0178] The assay for centrifugal separation of plasma components wasbased on Rudel Biochem J., 139, 89-95, 1974.) and subsequently modifiedby Alison et el. Photochem. Photobiol. 52(3): 501-507, 1990). It hasbeen scaled down to allow a shorter centrifugation time. Evidence ofclear separation and identities of the different layers has beenestablished. MACE (monoaspartyl chlorin e6) is a relatively watersoluble photosensitizer known to be bound and transported by albumin inthe circulation. The validity of this assay was further tested usingMACE, which was found to be overwhelmingly associated with the albumin(87%), with very little in the lipoprotein layer (11%).

[0179] Fresh human plasma was collected in EDTA, and KBr added to give aconcentration of 1.21-1.23 g/ml. Photosensitizer formulations were addedto 0.8 ml pre-warmed plasma (37° C.) to give a final concentration of 10μg/ml. 30 sec later, plasma was cooled for 15 min on ice, and underlayered with 2.45 ml KBr/EDTA at 1.21 g/ml in thick polycarbonate tubes.Samples were centrifuged at 512K g (100,000 RPM, Beckman TLA 100.3rotor) for 16-18 h at 20° C. Layer positions were marked to allowdetermination of layer volume. Each layer was sampled by removing aportion using a syringe inserted from the top. Known volumes of plasmalayers were removed into TX/PBS in an 1.8 ml tube (Eppendorf Scientific,Inc., Eppendorf) to give a final concentration of 1% TX. Samples werevortex mixed and then spun for 2 min at 14 000 RPM in an Eppendorfcentrifuge for clarification. Fluorescence at 690 nm (λ_(ex)=434 nm) wasread alongside standards of known drug concentration. Total drug presentin each layer was calculated on the basis of known layer volume andabsorbance value.

[0180] Tables 9 and 10 show the percentage distribution of B-B3 andB-EA6, in the various components of the fractionated plasma incomparison to BPD-MA, using liposomal, copolymer and DMSO formulations.

[0181] As expected from previous studied liposomal BPD-MA associatedpredominantly with the lipoproteins (Tables 9 and 10). Comparableresults were obtained for the liposomal B-EA6 formulation (Table 9) butnot for liposomal B-B3 (Table 10). Surprisingly, the copolymerformulation of B-B3 was found to be superior for delivering the B-B3 tothe lipoprotein fraction compared to the liposomal formulation (Table9). Delivery of the B-EA6 was comparable to the liposomal formulation.The results also showed that delivery of both liposomal and copolymerformulation of EA6-B and B3-B to the lipoprotein fraction was moreefficient than with DMSO formulations. TABLE 9 Percent B-B3 associatedwith various plasma fractions following centrifugal separation LiposomalLiposomal P123 DMSO BPD-MA B-B3 B-B3 B-B3 Plasma % % % % Band Component(n = 4) (n = 2) (n = 6) (n = 2) A Lipoprotein 85.0 (3.6)¹ 61.4 (1.76)91.8 (1.2) 61.2 (1.12) B′ Salt water  5.8 (1.4)  9.4 (0.42)  4.6 (1.3)15.0 (0.21) C′ Albumin  6.5 (2.3)   23 (1.51)  0.8 (0.1)  1.9 (0.65) COther  0.6 (0.2)  1.4 (0.01)  0.4 (0.2)  4.6 (0.23) proteins X Pellet 2.1 (0.8)  4.8 (0.16)  2.4 (0.2) 17.4 (0.47) Average 79.75 95.55 103.0376.1 Recovery

[0182] TABLE 10 Percent B-EA6 associated with various plasma fractionsfollowing centrifugal separation Liposomal Liposomal P123 DMSO DMSOBPD-MA B-EA6 B-EA6 B-EA6 BPD-MA Plasma % % % % % Band Component (n = 4)(n = 2) (n = 6) (n = 2) (n = 2) A Lipoprotein 85.1 (2.8)¹ 89.4 (0.04)91.4 (2.3) 59.0 (1.44) 74.0 (2.3) B′ Salt water  6.8 (1.0)  8.5 (0.04) 3.5 (1.3) 14.6 (1.10) 15.7 (1.8) C′ Albumin  6.9 (1.7)  0.8 (0.10)  1.5(0.6)  2.8 (0.04)  6.0 (0.3) C Other proteins  0.5 (0.2)  0.4 (0.01) 0.2 (0.2)  2.6 (0.09)  2.8 (0.4) X Pellet  0.7 (0.4)  0.9 (0.01)  4.2(1.8) 21.0 (2.45)  1.4 (0.5) Average 92.05 90.8 87.17 77.95 84.2Recovery

[0183] Addition of BPD-MA/DMSO to plasma resulted in inefficientdelivery to the lipoprotein fraction in comparison to the liposomalformulation. All drugs added to plasma in DMSO resulted in high drugconcentration in the salt/water fraction and in the pellet. Althoughthere appears to be a genuine binding to the sedimented flocculent, drugaggregates also end up in the pellet. Low total drug recoveries wereobserved in DMSO formulations, which probably reflects inadequatedissociation of these aggregates in the detergent system used to readassays.

[0184] The above example demonstrates that the copolymer formulations ofB-ring compounds are either comparable or superior to the liposomalformulations for the delivery of the drug to the lipoprotein fraction ofthe plasma. This is important for PDT because most target tissues, thoseundergoing rapid proliferation or repair, express high levels of LDLreceptors, and lipoprotein mediated delivery results in selectiveaccumulation of photosensitizers in these tissues.

Example 10 Cellular Uptake of Liposomal and Polymer Delivery ofPhotosensitizers

[0185] The following example illustrates the efficiency of cellularuptake using block copolymer formulation of a B-ring photosensitizer,B-B3, in comparison with the standard liposomal formulation of BPD-MA.

[0186] For this experiment the B-B3 copolymeric formulation and theBPD-MA liposomal formulation were prepared as described in Examples 5and 6, respectively. The protocol for setting up the cell cultures andconditions for the cellular assay essentially followed Richter et al.(Proc. SPIE, 2078: 293-304, September 1993). L1210 cells in DMEM and 10%FBS (single experiment, 3 sets) were incubated with the formulations ata concentration of 3 μg/ml and examined for uptake in the cells overtime. Cells were recovered by centrifugation, the pellet briefly rinsed,and the cells lysed by freeze thawing in the presence of 2% TritonX-100®. An equal volume of methanol was added and fluorescence was readat 694 nm (λ_(ex) 440 nm).

[0187]FIG. 1 shows that cellular uptake of the B-B3 copolymerformulation was very rapid compared to BPD-MA liposomal formulation. 50%uptake level was observed to be close to ‘zero’ incubation time, withuptake of B-B3 peaking at around 20 min. In comparison, BPD-MA achievedsaturation level at 30 min, with 50% uptake at approximately 5 min. Itappears that the permeability of cellular membranes to B-B3 is higher inthe presence of P123. This is important for the effective penetration ofthe photosensitizer into the PDT sensitive sites in the intracellularinfra structure.

[0188] These results suggests that light exposure for PDT treatment ingeneral could be applied as early as 10 to 15 min post injection if thephotosensitizer is formulated in copolymers.

[0189] The above example demonstrates rapid uptake of a B-Ringphotosensitizer by cells when using copolymer. Further because of therapidity of the photosensitizer uptake using copolymer formulation bythe targeted cells, the irradiation step for PDT can be carried outearlier than previously reported for liposomal or other formulations.

Example 11 Comparison of Block Copolymer and Liposomal PhotosensitizerFormulations: in vitro Phototoxicity

[0190] The following example illustrates the advantages of usingPluronic based formulations for effective delivery of B-ringphotosensitizer drugs to the cells in a model system.

[0191] In this experiment copolymer P123, liposomal and DMSOformulations of the B-ring compounds, B-EA6 and B-B3, were examined fortheir in vitro cytotoxicity effects. Exposure to drugs was carried outin the presence and absence of fetal calf serum (FCS) as a model tostudy transfer of drug to cells in vivo. BPD-MA liposomal formulationwas used as the standard and the DMSO formulation as the control. TheDMSO, Pluronic micellar and liposomal formulations of thephotosensitizers were prepared as described in Example 9. A suspensionof L1210 cells was prepared and exposed to various drug formulations(drug concentrations ranging from 0-50 ng/ml) either in the absence orpresence of 10% fetal calf serum (FCS). One hour later, the drug wasremoved by pelleting the cells by centrifugation. The pellet was brieflywashed with 1 ml DME and resuspended in 5% FCS/DME. 100 μl of the cellsuspension was aliquoted into 6 wells of a 96 well plate, and the plateexposed to light at 10 J/cm². Viability was determined 24 h postexposure using the MTT assay (Mosmann, J. Immunol.Meth. 65:55-63, 1983).TABLE 11 LD₅₀ (ng/ml) Photosensitizer Carrier −FCS +FCS BPD-MA Liposomal4.0 38.0 B-B3 Copolymer 0.68* 16.6* B-B3 Liposomal 3.0 30.0 B-B3 DMSO7.2 37.0 B-EA6 Copolymer 2.06* 12.9* B-EA6 Liposomal 4.7 19.7 B-EA6 DMSO4.7 20.0

[0192] Table 11 shows the LD₅₀ values determined for in vitrophotocytotoxicity for formulations of B-ring drugs in block copolymerscompared to drug delivery using liposomes and solutions in DMSO.

[0193] The presence of FCS better represents in vivo conditions forcellular exposure to systemic drugs, and under these conditions itgenerally competes with the cells for drug binding. However, under bothconditions, it is clear from the LD₅₀ values that formulations of B-ringdrugs in Pluronic have greater potency than liposomal formulations orsolutions in DMSO. This indicates superior delivery of drug in anon-aggregated form to cells or plasma proteins. Without being bound bytheory, the advantage could also be partly attributed topermeabilization of cellular membranes by poloxamers, which would allowbetter access of the drug to PDT-sensitive intracellular sites.

[0194] The above example demonstrates that the B-ring compoundsformulated with P123 were successfully delivered to the cells in anon-aggregated form. The delivery of the photosensitizer drug with thecopolymer formulation was found to be superior to the liposomalformulations.

Example 12 Comparison of B-B3 Copolymer and Liposomal Formulations forPDT Treatment of Arthritis in MRL/lpr Mouse Model

[0195] Arthritis in the MRL/lpr mouse strain was enhanced by giving 2intradermal injections (thoracic and inguinal sites) with 0.05 ml ofcomplete Freunds adjuvant containing 10 mg/ml heat-inactivated M.tuberculosis. PDT was given on days 0, 10 and 20 following CFAtreatment. PDT was carried out as follows; 3 groups of MRL/lpr mice wereinjected intravenously with B-B3 at 0.5 mg/kg (copolymer or liposomalformulations), after which they were protected from light. The thirdgroup was injected with copolymer alone at an equivalent copolymerconcentration to that found in the formulation. An hour later, they wereexposed to light at 80 J/cm² for 1.5 h (8 mW/cm²).

[0196] Ankle width measurements were taken every 5 days prior to PDTtreatment. The results of the above experiment are shown in FIG. 2. Micereceiving copolymer alone exhibited symptoms similar to the untreatedcontrol. The liposomal formulation of B-B3 in earlier part of the studyshowed better suppression of the inflammation compared to the controls.However, after day 25 there was an exacerbation of the inflammatorycondition. Relative to the controls and the liposomal formulation, theB-B3 copolymer formulation was highly effective in controlling theinflammation as determined by increase in ankle swelling.

[0197] The above example demonstrates that copolymer formulation of B-B3is superior to the liposomal formula for controlling an inflammatorydisease in vivo in arthritic mouse model.

Example 13 Optimization of B-B3 Intravenous Formulation in Pluronic P123

[0198] The following example illustrates the effects of copolymer:drugratio in achieving total drug incorporation.

[0199] Using formulation methods described in Example 5, the aim was toincorporate 2 mg/ml of B-B3 into 10% w/v P123. It was shown by thismethod that the B-B3 can typically be incorporated at ˜1.8 mg/ml drug(based on absorbance readings and a molar extinction coefficient of30425) 24 h post-hydration. This translates to approximately 10% drugloss. Unincorporated drug undergoes aggregation in aqueous solutions,and is characterized by the appearance of a 730 nm absorbance peak.Although the formulations can be made completely aggregate free bycentrifugation or sterile filtration through 0.2 μm filters, this addsanother step in the manufacturing process, which can be avoided byincreasing the copolymer:drug ratio. A final drug concentration of 1mg/ml resulted in complete incorporation of all added drug.

Example 14 Blending of Copolymers for Intravenous Formulations of B-B3

[0200] To achieve a solid final product, the hydrated material islyophilized. Alternative means of drying include, but is not limited to,spray or freeze drying. It is important to determine whether the dryingprocess affects the product integrity and to ascertain that formulationcharacteristics are retained on reconstitution.

[0201] In this experiment a 10% P123 (w/v) resulted in a thin film, withan oily appearance, which was difficult to hydrate. Counteracting theoily nature of P123 could be achieved by incorporation of copolymer thatis in solid form at room temperature. The use of 1% w/w F127 with 9% w/vP123 instead of 10% P123 (w/v) produced a thin film, which was morereadily hydrated. This composition was equally stable and was readilyreconstituted following lyophilization. The use of blends may be used totailor a formulation according to the needs of the particular drugsubstance and/or to compensate for properties lacking in a primarycopolymer.

[0202] pH studies showed that acidification of B-B3 formulations wasdetrimental to formulation stability. This necessitates hydration of thesolid drug-polymer with a very mild buffer to counteract acidificationwhich occurs upon use of sterile packed distilled water as commonlypracticed in clinical settings. Behavior of poloxamers is unaffected bypH, and the use of buffers would be entirely dependent on ionizablegroups present on the drug substance. For example, B-EA6 does notdisplay any pH-dependency.

[0203] The above example demonstrates that using blend of copolymers forformulating photosensitizer improved the rehydration of thephotosensitizer after lyophilization. It also shows that only mildbuffers are needed since the copolymer is unaffected by pH, unlikeliposomes.

Example 15 Deposition of Block Copolymer Photosensitizer BasedFormulations on Sugar Crystals

[0204] This example demonstrates that the use of the micro thin film canbe extended beyond lipids to any alternative carriers for hydrophobicphotosensitizer drugs. The use of the micro-thin film technique forformulation of photosensitizer drug using block copolymer and depositionon sugar crystals resulted in a solid-state formulation that is easy tohydrate.

[0205] In this experiment the deposition of the photosensitizer BPD-MAwith the block copolymer Pluronic® F127 onto the sugar lactose wasexamined. Formulations containing 5%(w/v) and a 10% (w/v) F127 weretested. 0.5 g lactose and 10 mg BPD-MA were added to two rotaryevaporation flasks. A stock solution of 0.2 mg/ml F127 was prepared inCH₂Cl₂. 1.25 ml (for 5% w/v) and 2.5 ml (for 10% w/v) F127 stocksolution was added to each flask. The final volume in each flask wasmade up to 5.0 ml with CH₂Cl₂ and the components mixed to ensurecomplete dissolution. The solvent was removed by rotary evacuation at50° C., and the flask left under vacuum for a further 15 min at 23° C.Micro-thin film deposits were scraped from the walls and hydrated in 5ml water at 50° C. The formulations were filtered twice using 0.2 μmsyringe filters (Acrodisc, polysulphone).

[0206] It was observed that both the thin film formulations dissolvedeasily, particularly 5% w/v, which went into solution immediately onaddition of water. Both the formulations (5% w/v and 10% w/v F127)filtered easily through 0.2 μm filters and with no drug loss.

[0207] The above example demonstrates that solid-state formulation of anA-ring photosensitizer and block copolymer carrier deposited on sugarcrystals offers a very simple alternative to liposomal-basedformulations. Furthermore, if prepared under sterile GMP conditions itcan provide a simple, one step manufacturing process.

Example 16 Deposition of Block Copolymer Photosensitizer BasedFormulations onto Sugar Crystals Using Ethanol as Solvent

[0208] This experiment examines the substitution of ethanol for CH₂Cl₂as the solvent for dissolving the block copolymer F127, andphotosensitizer BPD-MA, for deposition on lactose crystals. It alsoexamined the use of lower concentration of F127 for the formulation.

[0209] The experimental conditions and components were the same asExample 15 with the exception of the following changes. A stock of 0.2mg/ml F127 was prepared in ethanol and 0.65 ml (2.5% w/v) and 1.25 ml(5% w/v) of the stock solution was added to two flasks. The final volumewas made up to 5.0 ml with ethanol and the contents dissolved withwarming. Ethanol was removed by rotary evacuation at 50° C., left undervacuum for 15 min at room. Micro-thin film deposits were scraped fromthe walls and dissolved in 5 ml water at 50° C. as previously described.Samples were filtered 3 times through 0.2 μm syringe filters.

[0210] Substitution of ethanol for CH₂Cl₂ as the solvent for dissolvingand depositing the formulation on lactose crystals was successful. Boththe 2.5% and 5% w/v of F127 formed micro-thin films after removal ofethanol, and were easily hydrated. Further these formulations werefiltered through 0.2 μm filter with no resistance.

[0211] The above example demonstrates that ethanol can replace CH₂Cl₂ asthe volatile solvent for dissolving block copolymer and A-ringphotosensitizer for deposition on lactose sugar crystals.

Example 17 B-Ring Photosensitizers Formulations Using Mixed BlockCopolymers

[0212] This example illustrates the of blended block copolymers fordissolving and improving the hydration of B-ring photosensitizer solidsupport based formulations.

[0213] The poloxamer that was found to be useful in formulating a rangeof tetrapyrrolic drugs was Pluronic® P123, under the above conditions.

[0214] In this experiment formulation of B-ring photosensitizer, B-B3 at2 mg/ml with blended P123 and F127 or PVP, using the thin film method asdescribed in Example 15 were examined. The aim of the followingexperiment was to determine whether incorporation of solid compounds(e.g., PVP, F127) into the formulation might help to counteract the waxynature of P123 in the thin film, hence improving hydrationcharacteristics without destabilizing the formulation.

[0215] The polymer combinations used in this experiment are described inthe following table. The relative ease of thin film hydration for eachcombination was observed. The drug concentration retention wasdetermined by absorbance at t=0, 3 h and 24 h. Following centrifugationeach sample was diluted to 1:100 dilution in MeOH and A690 measured.

[0216] The relative ease of hydration for the poloxamer or poloxamercombinations was observed to be as follows:

[0217] 5% P123+5% F127>5% P123+5%PVP>10% P123+5%PVP>10% P123

[0218] P123 is semi solid and its waxy in nature makes it very difficultto hydrate. Based on the above results, formulations with a lower P123content hydrated more readily. The presence of solid compounds such asPVP and F127 in combination with P123 facilitated the hydration of theformulations. Incorporation of crystalline lactose is advantageousbecause it resulted in the improvement of the quality of the thin film,which was drier and thinner and therefore easier to hydrate, compared tothe previous poloxamer based thin films, which were then hydrated withiso-osmolar lactose solution.

[0219] The result of the drug retention measurement over time is shownin Table 12. TABLE 12 B-B3 Retention In Various Polymeric FormulationDetermined By Absorbance Readings (690 nm) A₆₉₀ Polymer Combination T =0 T = 3 h T = 24 h 5% P123 + 5% F127 0.88 0.54 0.42 0.93 0.57 0.45 5%P123 + 5% PVP 0.84 0.75 0.57 0.91 0.75 0.61 10% P123 + 5% PVP 0.88 0.690.45 0.89 0.71 0.44 10% P123 0.77 0.91 0.84 0.81 0.91 0.79

[0220] The results show that all samples formulated in blended polymerslose drug on standing over 24 hours. It was observed that 10% P123retained the most drug. The drug retention in the formulation after 24 hwas in the following order:

[0221] 10% P123>10% P123+5% PVP>5% P123+5% PVP>5% P123+5% F127

[0222] These results indicate that the presence of P123 in theformulation allows for B-EA6 drug to be stable in the formulation. Ithas been previously shown that drug formulation with 10% w/v F127resulted in poor formulation efficiency for B-EA6 (see Example 4 above).The use of various molecular weights of PVPs with the photosensitizerBPD-MA, also resulted in poor retention of the drug (results not shown).

[0223] The above example demonstrates that B-ring photosensitizer drugformulation and hydration is improved with blending of polymers and useof lactose. Pluronic P123, a block copolymer that is semi-solid and waxyat ambient temperatures, when blended with PVP or other blockcopolymers, such as Pluronic F127, which are solids, was shown toimproves hydration of B-EA6 thin film preparation.

Example 18 Photosensitizers Formulations Using Mixed Block Copolymersand Dissolvable Crystalline Solid Support

[0224] The objective of this experiment was to optimize thephotosensitizer drug stability using different blends of copolymercontent in the formulation while retaining the ease of hydration of thesugar based thin film. The effect of lyophilization of hydrated materialwas also examined.

[0225] Initially the aim was to incorporate 2 mg/ml of B-B3 into 10% w/vP123 by this method. It was shown in previous work that ˜1.8 mg/ml B-B3can typically be retained 24 h post-hydration. This translates toapproximately 10% drug loss. Unincorporated B-ring drugs undergoaggregation in aqueous solutions, which is characterized by appearanceof a 730 nm absorbance peak. Although the formulations can be madecompletely aggregate free by centrifugation or sterile filtrationthrough 0.2 μm filters, this adds another step in the manufacturingprocess, which can be avoided by increasing the copolymer:drug ratio.

[0226] In this experiment the B3-B was formulated using the sugartrehalose (9.5% w/v) to give a final drug concentration of 1 mg/mL. Thenon-blended and blended poloxamer contents of the test samples were asfollows: 7.5% w/v P123; 9% w/v P123+1% w/v F127; and 10% w/v P123.

[0227] B-B3 was dissolved in CH₂Cl₂ to a concentration of 1 mg/mL, and 1mL of the solution was dispensed into 25 ml round bottom flasks. A 100mg/mL stock solution of Pluronic P123 in CH₂Cl₂ was prepared, anddispensed into the flasks, followed by solid F127 to give 7.5% w/v P123;9% w/v P123+1% w/v F127; and 10% w/v P123, in duplicate. Trehalose wasadded to give 9.5% w/v final concentration in each of the flasks.Solvent was removed by rotary evaporation to give a micro-thin filmcomposed of B3-B and copolymers deposited on trehalose crystals. Thefilms were hydrated with distilled water (adjusted to pH 7.6) at roomtemperature. Hydrated samples were studied for stability at roomtemperature for up to 24 h by spectroscopic scanning between 650 and 750nm following 1:100 dilution in water, pH 7.6. After 24 h stabilitystudies, samples were lyophilized at −10° C.

[0228] The relative ease of reconstitution of the lyophilizedformulations of the B-B3 with the various poloxamer combinationsdeposited on trehalose was observed to be as follows:

[0229] 7.5% P123>9% P123+1%F127>10% P123 TABLE 13 Dependence offormulation stability on block copolymer content Lyophilized FormulationA₆₉₀ Post Reconstitution (4 h) 7.5% P123 0.308 0.299 1% F127 + 9% P1230.332 0.382 10% P123 0.351 0.342

[0230] These results once again suggests that the lower the content ofthe waxy copolymer (e.g. Pluronic P123), the greater the ease ofhydration. In the previous example (Example 17) addition of 5% w/v solidcopolymer (F127) into P123 was shown to cause destabilization of theformulation, however in the present experiment incorporation of 1% w/vresulted in superior hydration of the micro-thin film, withoutcompromising formulation (Table 13).

[0231] The above example demonstrates that photosensitizers usingblended poloxamers as carriers and depositing onto sugar results instable solid-state formulations that are easier to hydrate, and retainthe photosensitizer drug in a non-aggregated form.

Example 19 Photosensitizers Formulations Using Mixed Block Copolymersand Soluble Crystalline Solid Supports

[0232] The following example demonstrates that complexes ofphotosensitizer drug blended copolymers P123 and F127 (lyophilizedmaterial) hydrate easier if trehalose is used as a solid support insteadof lactose.

[0233] This experiment examined the use of blended block copolymers, 9%P123 and 1% F127 with 9.5% w/v lactose or trehalose, as solid supportsfor formulating 1 mg/ml B-B3. The control was 10% P123 with either 9.5%w/v lactose or trehalose. The procedure was carried out as described inExample 18 and the hydration of the thin film, or ease of reconstitutionof the lyophilized preparations were examined. Formulations of B-B3 (1mg/mL) with copolymer content of 10% P123 and 9% P123+1% F127 wereprepared for comparison. Thin films were hydrated with 0.01Mcitrate-phosphate buffer pH 7.4. 1 mL of hydrated formulations wasaliquoted into 2 mL lyophilization vials and lyophilized.

[0234] All the samples formed lyophilized cakes that were observed to befluffy and -uniform in appearance. The ease of hydration of lyophilizedcakes were as follows:

[0235] 9% P123+1% F127+trehalose>10% P123+trehalose>9% P123+1%F127+lactose>10% P123+lactose

[0236] Although all B-B3 formulation samples formed cakes uponlyophilization, formulations containing trehalose were relatively easierto reconstitute compared to lactose based formulations. This wasirrespective of copolymer content. It was also confirmed that additionof solid copolymer, F127 to a concentration of 1% w/v resulted in easierreconstitution of the lyophilized cakes for both trehalose and lactosecontaining formulations.

Example 20 Comparison of Tumor Recurrence in Mice Model Treated with PDTUsing A- & B-Ring Photosensitizers in Block Copolymer and LiposomalFormulations

[0237] The following example illustrates that the efficiency ofpoloxamer based photosensitizer formulations over liposomal formulationin a tumor mouse model following PDT.

[0238] Photosensitizer formulations were prepared in 10% w/v Pluronicsas described in Example 4. BPD-MA was formulated in F127. B-ringcompounds B-EA6 and B-B3, were prepared in P123 due to insufficient drugloading in F127. Liposomal BPD-MA formulation was Verteporfin™ andB-ring compounds were formulated in the same lipid composition. WhereDMSO/plasma preparations were made, the DMSO dissolved drug was addeddirectly to mouse plasma and the drug association with different plasmacomponents was observed.

[0239] In these experiments the tumor model used was the DBA/2 mouse(males) inoculated intradermally with M1 rhabdomyosarcoma tumor cells(M1, ATCC. When tumors reached a diameter of 4-6 mm, the mice (n10,unless other wise stated) were treated with photodynamic therapy (PDT).PDT involved intravenous injection of the formulated drug in 0.2 mLvolume of PBS. This was followed by exposure of the tumor region tolaser light (Argon pumped dye laser (5W), 690 nm, 50J/cm²) 15 min later.Animals were then monitored for tumor recurrence over a 20 day periodpost treatment. TABLE 14 Results of Tumor Cure Following Administrationof Poloxamer Formulations; Comparison to Liposomal BPD-MAPhotosensitizer Percent (%) Photosensitizer Photosensitizer Mice TumorFree Formulation Type Dosage Day 7 Day 14 Day 20 BPD-MA Liposomal 1.0mg/kg 100 100 30 Pluronic F127 1.0 mg/kg 100  60 60 B-EA6 Liposomal 1.0mg/kg  90  70 60 Pluronic P123 1.0 mg/kg PT¹ PT¹ PT¹ Pluronic P123 0.5mg/kg  80  60 40 B-B3 Liposomal 1.0 mg/kg  0² —² —² Pluronic P123 1.0mg/kg 100  80 60 Pluronic P123 1.2 mg/kg 100³  67³ 67³ Pluronic P123 1.25 mg/kg 100 100 80

[0240] Table 14 summarizes the result of the above experiments. Theperformance of B-ring compounds was compared to the liposomal BPD-MA(Verteporfin) formulation which was used as the standard for assessingperformance of other photosensitizers and formulations. It was observedthat at the end of the 20 day period, mice treated with the poloxamerformulation were twice as likely to remain tumor free compared to thosetreated with liposomal BPD-MA.

[0241] Although B-EA6 formulated poorly in liposomes (in terms of drugloading), it demonstrated the greatest potency of the three liposomaldrugs tested in the mouse tumor model. Administration of the 1 mg/kgB-EA6, formulated in P123, to tumor bearing mice resulted in a strongphototoxic reaction (edema) at the irradiated site, and the animals wereconsequently euthanized. This observation suggested that better drugdelivery is achieved using poloxamers compared to the liposomalformulations at the same drug dosage. At a lower dose of 0.5 mg/kg, acure rate was achieved similar to that of liposomal formulations ofB-EA6 and BPD-MA at 1 mg/kg.

[0242] B-B3 demonstrated greatest sensitivity to the drug delivery agent(or “carrier”) used in the formulation. At these levels, the plasmaIDMSO preparation was found to be completely ineffective for PDTpurposes. One of the most important modes of action of PDT is thedisruption of neovasculature. Performance of B-B3 formulated in P123 at1 mg/ml was marginally better than that of liposomal BPD-MA, andcomparable to BPD-MA in poloxamer formulations. Increasing the dose B-B3by 25% resulted in a marked improvement in performance in the tumorassay.

[0243] The results show that B-ring compounds formulated in poloxamerssuch Pluronic P123 enhanced performance of PDT in vivo. Without beingbound by theory, the observed effects could be attributed partly tofacilitation of the drugs across cellular membranes by the poloxamer andpartly to improved delivery of drug to plasma lipoproteins. Althoughboth B-ring compounds EA6 and B-3 had a tendency to aggregate, it wasthe amount associated with the lipoprotein fraction that dictated theefficacy of PDT in vivo. B-B3 showed poor delivery to the lipoproteinfraction for both liposomal and DMSO/plasma formulations (Table 9) andthis resulted in failure of PDT in the tumor model. On the other hand,in the case of liposomal and Pluronic formulation of B-EA6, delivery tolipoproteins was equivalent (Table 10), the results in vivo were notmarkedly different.

[0244] Furthermore, when comparing liposomal and poloxamer formulationsof B ring compounds, a lower concentration of the photosensitizer in thepoloxamer formulations appears to give similar results to those in theliposomal preparations. In fact, excessive photosensitivity at theirradiated site when using B-EA6 at the dose traditionally used forliposomal BPD-MA suggests that the drug dosage for achieving good PDTresponse can be considerably lowered. The above example demonstratesthat block copolymers allow formulation and potential use of B-ringcompounds (which were found ineffective or difficult to formulate inliposomes or homopolymers) to give photosensitizer products with greatlyenhanced drug delivery characteristics.

[0245] All references cited herein, including patents, patentapplications, and publications, are hereby incorporated by reference intheir entireties, whether previously specifically incorporated or not.As used herein, the terms “a”, “an”, and “any” are each intended toinclude both the singular and plural forms.

[0246] Having now fully described this invention, it will be appreciatedby those skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

[0247] While this invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications. This application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

We claim:
 1. A photosensitizer carrier composition comprising (a) one ormore photosensitizer and (b) one or more block copolymer in liquid formcapable of forming a complex with said photosensitizer and wherein saidcopolymer is i) not an amphiphilic polymer of polystyrene sodiumsulphonate and vinyl naphthalene, and ii) not poloxamer
 188. 2. A methodfor formulating a photosensitizer carrier composition of claim 1comprising combining one or more photosensitizer and one or more blockcopolymer in solution, wherein said copolymer is i) not an amphiphilicpolymer of polystyrene sodium sulphonate and vinyl naphthalene, and ii)not poloxamer
 188. 3. The method of claim 2 further comprising the stepof optionally drying said photosensitizer and copolymer combination. 4.A method for formulating a photosensitizer carrier composition accordingto claim 1 comprising combining one or more photosensitizer and one ormore block copolymer wherein said copolymer is in a liquefied state,said photosensitizer is soluble in said copolymer, and said copolymer isi) not an amphiphilic polymer of polystyrene sodium sulphonate and vinylnaphthalene, and ii) not poloxamer
 188. 5. The method of claim 3 furthercomprising the step of optionally hydrating said photosensitizer andcopolymer combination to form a complex.
 6. A method for conductingphotodynamic therapy comprising: (a) administering a photosensitizer andcopolymer complex produced by hydration of the composition of claim 1 toa subject in need of photodynamic therapy; and (b) irradiating saidsubject to activate said photosensitizer; wherein said copolymer is i)not an amphiphilic polymer of polystyrene sodium sulphonate and vinylnaphthalene, and ii) not poloxamer
 188. 7. The composition of claim 1,wherein said complex is selected from the group consisting of micelles,emulsions, gels, matrix or transition phases between the precedingdefined states.
 8. The composition of claim 1, wherein said blockcopolymer comprises polyoxyethylene and polyoxypropylene;polytetrahydrofuran; or polyaspartic acid.
 9. The composition of claim8, wherein said polyoxyethylene-polyoxypropylene block copolymer isselected from the group consisting of poloxamer 403 (P123), poloxamer407 (F127), poloxamer 402 (L122), poloxamer 181 (L61), poloxamer 401(L121), poloxamer 185 (P65), and poloxamer 338 (F108).
 10. Thecomposition of claim 1, wherein said photosensitizer is selected fromthe group consisting of porphyrins, pyrroles, tetrapyrrolic compounds,expanded pyrrolic macrocycles and their derivatives.
 11. The compositionof claim 10, wherein said porphyrin derivative is selected from thegroup consisting of green porphyrins, tetrahydrochlorins,pyrophenophorphides, purpurins, texaphyrins, phenothiaziniums,phthalocyanines, napthalocyanines, porphycenes and pheophorbides. 12.The composition of claim 11, wherein said tetrahydrochlorins areselected from the group consisting of chlorins, hydroxychlorins,bacteriochlorins, and isobacteriochlorins.
 13. The composition of claim12, wherein said green porphyrin is selected a benzoporphyrin derivative(BPD).
 14. The composition of claim 13, wherein said BPD is selectedfrom the group consisting of BPD-MA, BPD-MB, A-EA6, B-EA6, A-B3 andB-B3.
 15. The method of claim 5 wherein said combining step furthercomprises a hydratable solid support on which said photosensitizer andblock copolymer combination may deposit.
 16. The method of claim 15,wherein said solid support is not capable of dissolving in saidliquefied copolymer.
 17. The method of claim 15, wherein said solidsupport is capable of dissolving in said optional hydrating step.
 18. Aphotosensitizer carrier composition comprising a photosensitizer,poloxamer 188 (F68) and an emulsion forming agent, wherein said agent isnot a fluorocarbon selected from the group consisting of FC43, PP11, andPP25.
 19. The composition of claim 18 wherein said agent is not afluorocarbon.
 20. A method for conducting photodynamic therapycomprising administering a photosensitizer and copolymer complexproduced by hydration of the composition of claim 18 to a subject inneed of photodynamic therapy, and irradiating said subject to activatesaid photosensitizer.